Alpha-isomaltosylglucosaccharide-forming enzyme, process and uses of the same

ABSTRACT

The object of the present invention is to provide an α-isomaltosylglucosaccharide-forming enzyme, process of the same, cyclotetrasaccharide, and saccharide composition comprising the saccharide which are obtainable by using the enzyme; and is solved by establishing an α-isomaltosylglucosaccharide-forming enzyme which forms a saccharide, having a glucose polymerization degree of at least three and having both the α-1,6 glucosidic linkage as a linkage at the non-reducing end and the α-1,4 glucosidic linkage other than the linkage at the non-reducing end, by catalyzing the α-glucosyl-transfer from a saccharide having a glucose polymerization degree of at least two and having the α-1,4 glucosidic linkage as a linkage at the non-reducing end without substantially increasing the reducing power; α-isomaltosyl-transferring method using the enzyme; method for forming α-isomaltosylglucosaccharide; process for producing a cyclotetrasaccharide having the structure of cyclo{66)-α-D-glucopyranosyl-(163)-α-D-glucopyranosyl-(166)-α-D-glucopyranosyl-(163)-α-D-glucopyranosyl-(16} using both the α-isomaltosylglucosaccharide-forming enzyme and the α-isomaltosyl-transferring enzyme; and the uses of the saccharides obtainable therewith.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a division of application Ser. No. 10/089,549, filed Apr. 1,2002, which is a 371 national stage application of PCT/JP01/06412, filedJul. 25, 2001. The entire contents of both applications beingincorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a novelα-isomaltosylglucosaccharide-forming enzyme, process and uses of thesame, more particularly, to a novel α-isomaltosylglucosaccharide-formingenzyme, process for producing the enzyme, α-glucosyl-transferring methodusing the enzyme, process for producing a cyclotetrasaccharide havingthe structure ofcyclo{66)-α-D-glucopyranosyl-(163)-α-D-glucopyranosyl-(166)-α-D-glucopyranosyl-(163)-α-D-glucopyranosyl-(16}using both the enzyme and an α-isomaltosyl-transferring enzyme, andcompositions comprising these saccharides.

BACKGROUND ART

There have been known saccharides composed of glucose molecules asconstituent saccharides, for example, partial starch hydrolysates,produced from starches as materials, including amylose, amylodextrin,maltodextrin, maltooligosaccharide, and isomaltooligosaccharide. Also,these saccharides are known to have usually reducing and non-reducinggroups at their molecular ends and exhibit reducibility. In general,partial starch hydrolysates can be expressed with an index of dextroseequivalent (DE), a scale of reducing power based on the dry solid. Thosewith a relatively high DE value are usually known to have properties ofa relatively low molecular weight, relatively low viscosity, strongsweetness and reactivity, easy reactivity with amino group-containingsubstances such as amino acids and proteins that may induce browning andunpleasant smell, and easily cause deterioration. To improve thesedefects, there has long been desired a method for lowering oreliminating the reducing power without altering glucose molecules asconstituent saccharides of partial starch hydrolysates. For example, asdisclosed in Journal of American Chemical Society, Vol. 71, pp. 353-358(1949), it was reported that a method for forming α-, β-, andγ-cyclodextrins that are composed of 6-8 glucose molecules linkedtogether via the α-1,4 glucosidic linkage by contacting amylases,derived from microorganisms of the species Bacillus macerans, withstarches. Nowadays, these cyclodextrins are produced on an industrialscale and used in diversified fields using their inherent propertiessuch as non-reducibility, tasteless, and enclosing ability. Asdisclosed, for example, in Japanese Patent Kokai Nos. 143,876/95 and213,283/95 applied for by the same applicant as the present invention,it is known a method for producing trehalose, composed of two glucosemolecules linked together via the α,α-linkage, by contacting anon-reducing saccharide-forming enzyme and a trehalose-releasing enzymewith partial starch hydrolysates such as maltooligosaccharide. Atpresent, trehalose has been industrially produced from starches and usedin different fields by using its advantageous non-reducibility, mild-and high quality-sweetness. As described above, trehalose having aglucose polymerization degree (DP) of two, and α-, β-, andγ-cyclodextrins having a DP of 6-8 are produced on an industrial scaleand used in view of their advantageous properties, however, the types ofnon- or low-reducing saccharides are limited, so that more diversifiedsaccharides other than these saccharides are greatly required.

Recently, a new type of cyclotetrasaccharide, composed of glucose units,was reported. European Journal of Biochemistry, Vol. 226, pp. 641-648(1994) shows that a cyclic tetrasaccharide having the structure ofcyclo{66)-α-D-glucopyranosyl-(163)-α-D-glucopyranosyl-(166)-α-D-glucopyranosyl-(163)-α-D-glucopyranosyl-(16}(may be called “cyclotetrasaccharide” throughout the specification) isformed by contacting a hydrolyzing enzyme, alternanase, with alternanlinked with glucose molecules via the alternating α-1,3 and α-1,6 bonds,followed by crystallization in the presence of methanol as an organicsolvent.

Cyclotetrasaccharide or a non-reducing saccharide having a cyclicstructure, exhibits an inclusion ability to stabilize volatile organiccompounds, and does not cause an amino carbonyl reaction, and thereforeit is expected to be used and processed with lesser fear of browning anddeterioration.

However, the material alternan and alternanase, which are needed forproducing the saccharide, are not easily obtainable, and themicroorganisms for their production are not easily available.

Under such conditions, the present inventors succeeded in producingcyclotetrasaccharide by contacting, as a material, a saccharide havingthe α-1,6 glucosidic linkage as a linkage of non-reducing end and havinga glucose polymerization degree of at least three (may be called“α-isomaltosylglucosaccharide” throughout the specification) with anα-isomaltosyl-transferring enzyme which specifically hydrolyzes theα-isomaltosyl moiety and the resting glucosylsaccharide moiety and thentransfers the α-isomaltosyl moiety to its acceptor to formcyclotetrasaccharide, as disclosed in Japanese Patent Application Nos.149,484/2000 and 229,557/2000. The α-isomaltosyl-transferring enzyme isan enzyme which forms cyclotetrasaccharide fromα-isomaltoglucosaccharide by α-isomaltosyl-transferring reaction and hasthe following physicochemical properties:

(1) Action

-   -   Forming cyclotetrasaccharide having the structure of        cyclo{66)-α-D-glucopyranosyl-(163)-α-D-glucopyranosyl-(166)-α-D-glucopyranosyl-(163)-α-D-glucopyranosyl-(16}        from a saccharide having a glucose polymerization degree of at        least three and having both the α-1,6 glucosidic linkage as a        linkage at the non-reducing end and the α-1,4 glucosidic linkage        other than the above linkage;

(2) Molecular Weight

-   -   Having a molecular weight of about 82,000 to about 136,000        daltons when determined on sodium dodecyl sulfate (SDS)        polyacrylamide gel electrophoresis (PAGE);

(3) Isoelectric Point (pI)

-   -   Having a pI of about 3.7 to about 8.3 when determined on        isoelectrophoresis using ampholine;

(4) Optimum Temperature

-   -   Having an optimum temperature of about 45° C. to about 50° C.        when incubated at a pH of 6.0 for 30 min;

(5) Optimum pH

-   -   Having an optimum pH of about 5.5 to about 6.5 when incubated at        35° C. for 30 min;

(6) Thermal Stability

-   -   Having a thermostable range at temperatures of about 45° C. or        lower when incubated at a pH of 6.0 for 60 min; and

(7) pH Stability

-   -   Having a stable pH range at about 3.6 to about 10.0 when        incubated at 4° C. for 24 hours.

Referring to the material saccharides for cyclotetrasaccharide, itshould desirably be produced from the abundant and low-cost starches,however, since α-isomaltosyl-transferring enzyme does not directly acton starches, the following procedure is actually employed: Starches arefirst converted into such an α-isomaltosylglucosaccharide having theabove specified structure, for example, relatively-low molecular weightisomaltooligosaccharides such as panose and isomaltosylmaltose, and thensubjected to the action of α-isomaltosyl-transferring enzyme to formcyclotetrasaccharide.

It was found that, when used panose as a material forcyclotetrasaccharide, the yield of the saccharide from the material isabout 44% to the material, based on the weight of the dry solid(d.s.b.). Similarly, in the case of using isomaltosylmaltose as amaterial, the yield of cyclotetrasaccharide is about 31%, d.s.b., whilein the case of using starches as a material, they should be contactedwith enzymes such as α-amylase, starch debranching enzyme, β-amylase,and α-glucosidase to form relatively-low molecular weightisomaltooligosaccharides including panose, and the yield ofcyclotetrasaccharide is quite as low as about 15%, d.s.b.

Although the actual production of cyclotetrasaccharide is feasible fromstarches even with such a low yield, the production cost may beincreased. Under these circumstances, it is desired to establish a novelmethod for producing cyclotetrasaccharide in a relatively high yieldusing easily available materials such as starches.

DISCLOSURE OF INVENTION

The object of the present invention is to provide anα-isomaltosylglucosaccharide-forming enzyme, process of the same,cyclotetrasaccharide obtainable therewith, saccharides comprisingcyclotetrasaccharide, and uses thereof.

To solve the above object, the present inventors widely screenedmicroorganisms, which produce a novel enzyme usable for preparingcyclotetrasaccharide from starches as a material in a relatively highyield, with an expectation of obtaining such an enzyme. As a result,they unexpectedly found that the microorganisms of the genus Bacillus orArthrobacter such as Bacillus globisporus C9 strain, Bacillusglobisporus C11 strain, Bacillus globisporus N75 strain, andArthrobacter globiformis A19 (hereinafter may be called “Strain C9”,“Strain C11”, “Strain N75”, and “Strain A19”), which were isolated fromsoils, that form both an α-isomaltosyl-transferring enzyme, as disclosedin Japanese Patent Application Nos. 149,484/2000 and 229,557/2000, and anovel α-isomaltosylglucosaccharide-forming enzyme, which has beenpursued by the present inventors. The present inventors accomplishedthis invention by firstly finding the fact that the yield ofcyclotetrasaccharide, aimed at by the present inventors, can be greatlyimproved by contacting relatively-low molecular weight glucosylsaccharides which include partial starch hydrolysates withα-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme; revealed the properties ofα-isomaltosylglucosaccharide-forming enzyme, and preparation method ofα-isomaltosylglucosaccharide-forming enzyme; α-glucosyl-transferringreaction using α-isomaltosylglucosaccharide-forming enzyme, process forproducing α-isomaltosylglucosaccharide; cyclotetrasaccharide orsaccharide compositions comprising cyclotetrasaccharide, obtainable byusing α-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme; and process for producing thesesaccharides. Also, the present inventors established food products,cosmetics, and pharmaceuticals which comprise cyclotetrasaccharide orsaccharide compositions comprising the cyclotetrasaccharide, and thusaccomplished this invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an elution pattern of a saccharide, obtained byα-isomaltosyl-transferring enzyme from a microorganism of the speciesBacillus globisporus C9 strain, when determined on high-performanceliquid chromatography.

FIG. 2 is a nuclear resonance spectrum (¹H-NMR) of cyclotetrasaccharide,obtained by the enzymatic reaction using α-isomaltosyl-transferringenzyme from a microorganism of the species Bacillus globisporus C9strain.

FIG. 3 is a nuclear resonance spectrum (¹³C-NMR) ofcyclotetrasaccharide, obtained by the enzymatic reaction usingα-isomaltosyl-transferring enzyme from a microorganism of the speciesBacillus globisporus C9 strain.

FIG. 5 shows the thermal influence on the enzymatic activity ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Bacillus globisporus C9 strain.

FIG. 6 shows the pH influence on the enzymatic activity ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Bacillus globisporus C9 strain.

FIG. 7 shows the thermal stability ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Bacillus globisporus C9 strain.

FIG. 8 shows the pH stability of α-isomaltosylglucosaccharide-formingenzyme from a microorganism of the species Bacillus globisporus C9strain.

FIG. 9 shows the thermal influence on the enzymatic activity ofα-isomaltosyl-transferring enzyme from a microorganism of the speciesBacillus globisporus C9 strain.

FIG. 10 shows the pH influence on the enzymatic activity ofα-isomaltosyl-transferring enzyme from a microorganism of the speciesBacillus globisporus C9 strain.

FIG. 11 shows the thermal stability of α-isomaltosyl-transferring enzymefrom a microorganism of the species Bacillus globisporus C9 strain.

FIG. 12 shows the pH stability of α-isomaltosyl-transferring enzyme froma microorganism of the species Bacillus globisporus C9 strain.

FIG. 13 shows the thermal influence on the enzymatic activity ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Bacillus globisporus C11 strain.

FIG. 14 shows the pH influence on α-isomaltosylglucosaccharide-formingenzyme from a microorganism of the species Bacillus globisporus C11strain.

FIG. 15 shows the thermal stability ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Bacillus globisporus C11 strain.

FIG. 16 shows the pH stability of α-isomaltosylglucosaccharide-formingenzyme from a microorganism of the species Bacillus globisporus C11strain.

FIG. 17 shows the thermal influence on the enzymatic activity ofα-isomaltosyl-transferring enzyme from a microorganism of the speciesBacillus globisporus C11 strain.

FIG. 18 shows the pH influence on the enzymatic activity ofα-isomaltosyl-transferring enzyme from a microorganism of the speciesBacillus globisporus C11 strain.

FIG. 19 shows the thermal stability of α-isomaltosyl-transferring enzymefrom a microorganism of the species Bacillus globisporus C11 strain.

FIG. 20 shows the pH stability of α-isomaltosyl-transferring enzyme froma microorganism of the species Bacillus globisporus C11 strain.

FIG. 21 shows the thermal influence on the enzymatic activity ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Bacillus globisporus N75 strain.

FIG. 22 shows the pH influence on the enzymatic activity ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Bacillus globisporus N75 strain.

FIG. 23 shows the thermal stability ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Bacillus globisporus N75 strain.

FIG. 24 shows the pH stability of α-isomaltosylglucosaccharide-formingenzyme from a microorganism of the species Bacillus globisporus N75strain.

FIG. 25 shows the thermal influence on the enzymatic activity ofα-isomaltosyl-transferring enzyme from a microorganism of the speciesBacillus globisporus N75 strain.

FIG. 26 shows the pH influence on the enzymatic activity ofα-isomaltosyl-transferring enzyme from a microorganism of the speciesBacillus globisporus N75 strain.

FIG. 27 shows the thermal stability of α-isomaltosyl-transferring enzymefrom a microorganism of the species Bacillus globisporus N75 strain.

FIG. 28 shows the pH stability of α-isomaltosyl-transferring enzyme froma microorganism of the species Bacillus globisporus N75 strain.

FIG. 29 shows the thermal influence on the enzymatic activity ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Arthrobacter globiformis A19 strain.

FIG. 30 shows the pH influence on the enzymatic activity ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Arthrobacter globiformis A19 strain.

FIG. 31 shows the thermal stability ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Arthrobacter globiformis A19 strain.

FIG. 32 shows the pH stability of α-isomaltosylglucosaccharide-formingenzyme from a microorganism of the species Arthrobacter globiformis A19strain.

FIG. 33 shows the thermal influence on the enzymatic activity ofα-isomaltosyl-transferring enzyme from a microorganism of the speciesArthrobacter globiformis A19 strain.

FIG. 34 shows the pH influence on the enzymatic activity ofα-isomaltosyl-transferring enzyme from a microorganism of the speciesArthrobacter globiformis A19 strain.

FIG. 35 shows the thermal stability of α-isomaltosyl-transferring enzymefrom a microorganism of the species Arthrobacter globiformis A19 strain.

FIG. 36 shows the pH stability of α-isomaltosyl-transferring enzyme froma microorganism of the species Arthrobacter globiformis A19 strain.

FIG. 37 shows the thermal influence on the enzymatic activity ofα-isomaltosyl-transferring enzyme from a microorganism of the speciesArthrobacter ramosus S1 strain.

FIG. 38 shows the pH influence on the enzymatic activity ofα-isomaltosyl-transferring enzyme from a microorganism of the speciesArthrobacter ramosus S1 strain.

FIG. 39 shows the thermal stability of α-isomaltosyl-transferring enzymefrom a microorganism of the species Arthrobacter ramosus S1 strain.

FIG. 40 shows the pH stability of α-isomaltosyl-transferring enzyme froma microorganism of the species Arthrobacter ramosus S1 strain.

FIG. 41 is a nuclear resonance spectrum (¹H-NMR) ofα-isomaltosylmaltotriose, obtained by the enzymatic reaction usingα-isomaltosylglucosaccharide-forming enzyme of the present invention.

FIG. 42 is a nuclear resonance spectrum (¹H-NMR) ofα-isomaltosylmaltotetraose, obtained by the enzymatic reaction usingα-isomaltosylglucosaccharide-forming enzyme of the present invention.

FIG. 43 is a nuclear resonance spectrum (¹³C-NMR) ofα-isomaltosylmaltotriose, obtained by the enzymatic reaction usingα-isomaltosylglucosaccharide-forming enzyme of the present invention.

FIG. 44 is a nuclear resonance spectrum (¹³C-NMR) ofα-isomaltosylmaltotetraose, obtained by the enzymatic reaction usingα-isomaltosylglucosaccharide-forming enzyme of the present invention.

FIG. 45 is a visualized intermediate picture, displayed on a screen, ofa microscopic photo for the cyclotetrasaccharide crystal, penta- tohexa-hydrate, of the present invention.

FIG. 46 is an x-ray diffraction spectrum for the cyclotetrasaccharide,penta- to hexa-hydrate, in a crystalline form, of the present invention,when determined on x-ray powder diffraction analysis.

FIG. 47 is a thermogravimetric curve for the cyclotetrasaccharide,penta- to hexa-hydrate, in a crystalline form, of the present invention,when determined on thermogravimetric analysis.

FIG. 48 is an x-ray diffraction spectrum for the cyclotetrasaccharide,monohydrate, in a crystalline form, of the present invention, whendetermined on x-ray powder diffraction analysis.

FIG. 49 is a thermogravimetric curve for the cyclotetrasaccharide,monohydrate, in a crystalline form, of the present invention, whendetermined on thermogravimetric analysis.

FIG. 50 is an x-ray diffraction spectrum for an anhydrous crystallinepowder of the cyclotetrasaccharide of the present invention, obtained bydrying in vacuo at 40° C., when determined on x-ray powder diffractionanalysis.

FIG. 51 is an x-ray diffraction spectrum for an anhydrous crystallinepowder of the cyclotetrasaccharide, penta- to hexa-hydrate, of thepresent invention, obtained by drying in vacuo at 120° C., whendetermined on x-ray powder diffraction analysis.

FIG. 52 is a thermogravimetric curve for the anhydrouscyclotetrasaccharide powder of the present invention, when determined onthermogravimetric analysis.

BEST MODE FOR CARRYING OUT THE INVENTION

The following are the identification results of Strain C9, Strain C11,Strain N75, and Strain A19, which produce the novelα-isomaltosylglucosaccharide-forming enzyme of the present invention.The identification tests were conducted in accordance with the methodsas described in “Biseibutsu-no-Bunrui-to-Dotei” (Classification andIdentification of Microorganisms), edited by Takeji Hasegawa, publishedby Japan Scientific Societies Press, Tokyo, Japan (1985).

<Strain C9>

<A. Morphology>

-   -   Characteristics of cells when incubated at 27° C. in nutrient        broth agar    -   Existing usually in a rod shape of 0.5-1.0×1.5-5 Φm,    -   Exhibiting no polymorphism,    -   Possessing motility,    -   Forming spherical spores at an intracellular end and swelled        sporangia, and    -   Gram stain, positive;        <B. Cultural Property>    -   (1) Characteristics of colonies formed when incubated at 27° C.        in nutrient broth agar plate;        -   Shape: Circular colony having a diameter of 1-2 mm after two            days incubation        -   Rim: Entire        -   Projection: Hemispherical shape        -   Gloss: Dull        -   Surface: Smooth        -   Color: Opaque and pale yellow    -   (2) Characteristics of colony formed when incubated at 27° C. in        nutrient broth agar plate;        -   Growth: Roughly medium        -   Shape: Radiative    -   (3) Characteristics of colony formed when stab cultured at        27° C. in nutrient broth agar plate;        -   Liquefying the agar plate.            <C. Physiological Properties>    -   (1) VP-test: Negative    -   (2) Indole formation: Negative    -   (3) Gas formation from nitric acid: Positive    -   (4) Hydrolysis of starch: Positive    -   (5) Formation of pigment: Forming no soluble pigment    -   (6) Urease: Positive    -   (7) Oxidase: Positive    -   (8) Catalase: Positive    -   (9) Growth conditions: Growing at a pH of 5.5-9.0 and a        temperature of 10-35° C.    -   (10) Oxygen requirements: Aerobic

(11) Utilization of carbon source and acid formation Carbon sourceUtilization Acid formation D-Glucose + + Glycerol + + Sucrose + +Lactose + +Note:The symbol “+” means yes or positive.

-   -   (12) Mol % guanine (G) plus cytosine (C) of DNA: 40%

These bacteriological properties were compared with those of knownmicroorganisms with reference to Bergey's Manual of SystematicBacteriology, Vol. 2 (1986). As a result, it was revealed that themicroorganism was identified with a microorganism of the speciesBacillus globisporus. The microorganism had a feature, not disclosed inany literature, of forming both α-isomaltosylglucosaccharide-formingenzyme, which produces α-isomaltosylglucosaccharide from partial starchhydrolyzates, and α-isomaltosyl-transferring enzyme which producescyclotetrasaccharide from α-isomaltosylglucosaccharide by transferringα-isomaltosyl residue.

Based on these results, the present inventors named this microorganism“Bacillus globisporus C9”, and deposited it on Apr. 25, 2000, inInternational Patent Organism Depositary National Institute of AdvancedIndustrial Science and Technology Tsukuba Central 6, 1-1, Higashi1-Chome Tsukuba-shi, Ibaraki-ken, 305-8566, Japan. The deposition of themicroorganism was accepted on the same day by the institute under theaccession number of FERM BP-7143.

<Strain C11>

<A. Morphology>

-   -   Characteristics of cells when incubated at 27° C. in nutrient        broth agar    -   Existing usually in a rod shape of 0.5-1.0×1.5-5 Φm,    -   Exhibiting no polymorphism,    -   Possessing motility,    -   Forming spherical spores at an intracellular end and swelled        sporangia, and    -   Gram stain, positive;        <B. Cultural Property>    -   (1) Characteristics of colonies formed when incubated at 27° C.        in nutrient broth agar plate;        -   Shape: Circular colony having a diameter of 1-2 mm after two            days incubation        -   Rim: Entire        -   Projection: Hemispherical shape        -   Gloss: Dull        -   Surface: Smooth        -   Color: Opaque and pale yellow    -   (2) Characteristics of colony formed when incubated at 27° C. in        nutrient broth agar plate;        -   Growth: Roughly medium        -   Shape: Radiative    -   (3) Characteristics of colony formed when stab cultured at        27° C. in nutrient broth agar plate;        -   Liquefying the agar plate            <C. Physiological Properties>    -   (1) VP-test: Negative    -   (2) Indole formation: Negative    -   (3) Gas formation from nitric acid: Positive    -   (4) Hydrolysis of starch: Positive    -   (5) Formation of pigment: Forming no soluble pigment    -   (6) Urease: Positive    -   (7) Oxidase: Positive    -   (8) Catalase: Positive    -   (9) Growth conditions: Growing at a pH of 5.5-9.0 and a        temperature of 10-35° C.    -   (10) Oxygen requirements: Aerobic

(11) Utilization of carbon source and acid formation Carbon sourceUtilization Acid formation D-Glucose + + Glycerol + + Sucrose + +Lactose + +Note:The symbol “+” means yes or positive.

-   -   (12) Mol % guanine (G) plus cytosine (C) of DNA: 39%

These bacteriological properties were compared with those of knownmicroorganisms with reference to Bergey's Manual of SystematicBacteriology, Vol. 2 (1986). As a result, it was revealed that themicroorganism was identified with a microorganism of the speciesBacillus globisporus. The microorganism had a feature, not disclosed inany literature, of forming both α-isomaltosylglucosaccharide-formingenzyme, which produces α-isomaltosylglucosaccharide from partial starchhydrolyzates, and α-isomaltosyl-transferring enzyme which producescyclotetrasaccharide from the α-isomaltosylglucosaccharide bytransferring α-isomaltosyl residue.

Based on these results, the present inventors named this microorganism“Bacillus globisporus C11”, and deposited it on Apr. 25, 2000, inInternational Patent Organism Depositary National Institute of AdvancedIndustrial Science and Technology Tsukuba Central 6, 1-1, Higashi1-Chome Tsukuba-shi, Ibaraki-ken, 305-8566, Japan. The deposition of themicroorganism was accepted on the same day by the institute under theaccession number of FERM BP-7144.

<Strain N75>

<A. Morphology>

-   -   Characteristics of cells when incubated at 27° C. in nutrient        broth agar    -   Existing usually in a rod form of 0.5-1.0×1.5-5 Φm,    -   Exhibiting no polymorphism,    -   Possessing motility,    -   Forming spherical spores at an intracellular end and swelled        sporangia, and    -   Gram stain, positive;        <B. Cultural Property>    -   (1) Characteristics of colonies formed when incubated at 27° C.        in nutrient broth agar plate;        -   Shape: Circular colony having a diameter of 1-2 mm after two            days incubation        -   Rim: Entire        -   Projection: Hemispherical shape        -   Gloss: Dull        -   Surface: Smooth        -   Color: Opaque and pale yellow    -   (2) Characteristics of colony formed when incubated at 27° C. in        nutrient broth agar plate;        -   Growth: Roughly medium        -   Shape: Radiative    -   (3) Characteristics of colony formed when stab cultured at        27° C. in nutrient broth agar plate;    -   Liquefying the agar plate        <C. Physiological Properties>    -   (1) VP-test: Negative    -   (2) Indole formation: Negative    -   (3) Gas formation from nitric acid: Positive    -   (4) Hydrolysis of starch: Positive    -   (5) Formation of pigment: Forming no soluble pigment    -   (6) Urease: Negative    -   (7) Oxidase: Positive    -   (8) Catalase: Positive    -   (9) Growth conditions: Growing at a pH of 5.7-9.0 and a        temperature of 10-35° C.    -   (10) Oxygen requirements: Aerobic

(11) Utilization of carbon source and acid formation Carbon sourceUtilization Acid formation D-Glucose + + Glycerol + + Sucrose + +Lactose + +Note:The symbol “+” means yes or positive.

-   -   (12) Mol % guanine (G) plus cytosine (C) of DNA: 40%

These bacteriological properties were compared with those of knownmicroorganisms with reference to Bergey's. Manual of SystematicBacteriology, Vol. 2 (1986). As a result, it was revealed that themicroorganism was identified with a microorganism of the speciesBacillus globisporus. The microorganism had a feature, not disclosed inany literature, of forming both α-isomaltosylglucosaccharide-formingenzyme, which produces α-isomaltosylglucosaccharide from partial starchhydrolyzates, and α-isomaltosyl-transferring enzyme which producescyclotetrasaccharide from the α-isomaltosylglucosaccharide bytransferring α-isomaltosyl residue.

Based on these results, the present inventors named this microorganism“Bacillus globisporus N75”, and deposited it on May 16, 2001, inInternational Patent Organism Depositary National Institute of AdvancedIndustrial Science and Technology Tsukuba Central 6, 1-1, Higashi1-Chome Tsukuba-shi, Ibaraki-ken, 305-8566, Japan. The deposition of themicroorganism was accepted on the same day by the institute under theaccession number of FERM BP-7591.

<Strain A19>

<A. Morphology>

-   -   (1) Characteristics of cells when incubated at 27° C. in        nutrient broth agar;        -   Existing usually in a rod form of 0.4-0.8×1.0-4.0 Φm        -   Exhibiting polymorphism,        -   Possessing no motility,        -   Forming no spore, and        -   Gram stain, positive;    -   (2) Characteristics of cells when incubated at 27° C. in EYG        agar plate;        -   Exhibiting a growth cycle of bacillus and cocci            <B. Cultural Property>    -   (1) Characteristics of colonies formed when incubated at 27° C.        in nutrient broth agar plate;        -   Shape: Circular colony having a diameter of 2-3 mm after one            day incubation        -   Rim: Entire        -   Projection: Hemispherical shape        -   Gloss: Dull        -   Surface: Smooth        -   Color: Opaque and pale yellow    -   (2) Characteristics of colony formed when incubated at 27° C. in        nutrient broth agar plate;        -   Growth: Roughly medium        -   Shape: Filamentous    -   (3) Characteristics of colony formed when stab cultured at        27° C. in nutrient broth agar plate;        -   Not liquefying the agar plate.            <C. Physiological Properties>    -   (1) Hydrolysis of starch: Negative    -   (2) Formation of pigment: Forming no soluble pigment    -   (3) Urease: Positive    -   (4) Oxidase: Positive    -   (5) Catalase: Positive    -   (6) Oxygen requirements: Aerobic    -   (7) Main diamino acid of cell wall: Lysine    -   (8) Peptidoglycan type of cell wall: Lysine-alanine    -   (9) N-acyl type of cell wall: Acetyl    -   (10) Sugar component of cell wall: Galactose, glucose, rhamnose,        and mannose    -   (11) Vitamin requirements: Negative    -   (12) Mol % guanine (G) plus cytosine (C) of DNA: 62%    -   (13) DNA-DNA homology: Having a 66.5% of DNA-DNA homology when        compared with Arthrobacter globiformis, ATCC 8010.

These bacteriological properties were compared with those of knownmicroorganisms with reference to Bergey's Manual of SystematicBacteriology, Vol. 2 (1986). As a result, it was revealed that themicroorganism was identified with a microorganism of the speciesBacillus globisporus. The microorganism had a feature, not disclosed inany literature, of forming both α-isomaltosylglucosaccharide-formingenzyme, which produces α-isomaltosylglucosaccharide from partial starchhydrolyzates, and α-isomaltosyl-transferring enzyme which producescyclotetrasaccharide from the α-isomaltosylglucosaccharide bytransferring α-isomaltosyl residue.

Based on these results, the present inventors named this microorganism“Arthrobacter globiformis A19”, and deposited it on May 16, 2001, inInternational Patent Organism Depositary National Institute of AdvancedIndustrial Science and Technology Tsukuba Central 6, 1-1, Higashi1-Chome Tsukuba-shi, Ibaraki-ken, 305-8566, Japan. The deposition of themicroorganism was accepted on the same day by the institute under theaccession number of FERM BP-7590.

In the present invention, any microorganisms of the genera Bacillus,Arthrobacter, and others, as well as their mutants can be appropriatelyused as long as they form α-isomaltosylglucosaccharide-forming enzyme.These microorganisms can be easily screened by using conventionalscreening methods for microorganisms with an index of thephysicochemical properties of the α-isomaltosylglucosaccharide-formingenzyme of the present invention.

An α-isomaltosyl-transferring enzyme derived from a microorganism of thegenus Arthrobacter (hereinafter may be called “Strain S1”), isolated bythe present inventors from a soil in Okayama-shi, Okayama, Japan, can beadvantageously used in the present invention as theα-isomaltosyl-transferring enzyme which produces cyclotetrasaccharidefrom α-isomaltosylglucosaccharide by transferring α-isomaltosyl residue.The identification tests were conducted in accordance with the methodsas described in “Biseibutsu-no-Bunrui-to-Dotei” (Classification andIdentification of Microorganisms), edited by Takeji Hasegawa, publishedby Japan Scientific Societies Press, Tokyo, Japan (1985).

<Strain S1>

<A. Morphology>

-   -   Characteristics of cells when incubated at 27° C. in nutrient        broth agar;    -   Existing usually in a rod form of 0.3-0.7×0.8-3.5 Φm    -   Exhibiting polymorphism    -   Possessing no motility    -   Forming no spore    -   Gram stain: Positive    -   (2) Characteristics of cells when incubated at 27° C. in EYG        agar plate;        -   Exhibiting a growth cycle of bacillus and cocci            <B.: Cultural Property>    -   (1) Characteristics of colonies formed when incubated at 27° C.        in nutrient broth agar plate;        -   Shape: Circular colony having a diameter of 2-3 mm after one            day incubation        -   Rim: Entire        -   Projection: Hemispherical shape        -   Gloss: Dull        -   Surface: Smooth        -   Color: Opaque and pale yellow    -   (2) Characteristics of colony formed when incubated at 27° C. in        nutrient broth agar plate;        -   Growth: Roughly medium        -   Shape: Filamentous    -   (3) Characteristics of colony formed when stab cultured at        27° C. in nutrient broth agar plate;        -   Not liquefying the agar plate.            <C. Physiological Properties>    -   (1) Hydrolysis of starch: Negative    -   (2) Formation of pigment: Forming no soluble pigment    -   (3) Urease: Positive    -   (4) Oxidase: Positive    -   (5) Catalase: Positive    -   (6) Oxygen requirements: Aerobic    -   (7) Main diamino acid of cell wall: Lysine    -   (8) Peptidoglycan type of cell wall: Lysine-alanine    -   (9) N-acyl type of cell wall: Acetyl    -   (10) Sugar component of cell wall: Galactose, glucose, rhamnose,        and mannose    -   (11) Vitamin requirements: Negative    -   (12) Mol % guanine (G) plus cytosine (C) of DNA: 65%    -   (13) DNA-DNA homology: Having 84.4% of DNA-DNA homology when        compared with Arthrobacter ramosus, ATCC 13727.

These bacteriological properties were compared with those of knownmicroorganisms with reference to Bergey's Manual of SystematicBacteriology, Vol. 2 (1986). As a result, it was revealed that themicroorganism was identified with a microorganism of the speciesArthrobacter ramosus. The microorganism had a feature, not disclosed inany literature, forming both α-isomaltosylglucosaccharide-formingenzyme, which produces α-isomaltosylglucosaccharide from partial starchhydrolyzates, and α-isomaltosyl-transferring enzyme which producescyclotetrasaccharide from the α-isomaltosylglucosaccharide bytransferring α-isomaltosyl residue.

Based on these results, the present inventors named this microorganism“Arthrobacter ramosus S1”, and deposited it on May 16, 2001, inInternational Patent Organism Depositary National Institute of AdvancedIndustrial Science and Technology Tsukuba Central 6, 1-1, Higashi1-Chome Tsukuba-shi, Ibaraki-ken, 305-8566, Japan. The deposition of themicroorganism was accepted on the same day by the institute under theaccession number of FERM BP-7592.

In the present invention, any mutant of Arthrobacter ramosus S1 can beappropriately used in the present invention as long as it formsα-isomaltosylglucosaccharide-forming enzyme. Such a mutant can be easilyscreened by conventional screening methods for microorganisms with anindex of the physicochemical properties of theα-isomaltosyl-transferring enzyme usable the present invention.

Any nutrient culture medium can be used in the invention as long as theabove-mentioned microorganisms can grow therein and produce theα-isomaltosylglucosaccharide-forming enzyme; synthetic- andnatural-nutrient culture media can be arbitrarily used. The carbonsources usable in the present invention are those which themicroorganisms assimilate for their growth: Examples such are starchesand phytoglycogen from plants; glycogen and pullulan from animals andmicroorganisms; saccharides such as glucose, fructose, lactose, sucrose,mannitol, sorbitol, and molasses; and organic acids such as citric acidand succinic acid. The concentrations of these carbon sources innutrient culture media are appropriately changed depending on theirkinds. The nitrogen sources usable in the present invention are, forexample, inorganic nitrogen compounds such as ammonium salts andnitrates; and organic nitrogen-containing substances such as urea, cornsteep liquor, casein, peptone, yeast extract, and beef extract. Theinorganic ingredients usable in the present invention are, for example,calcium salts, magnesium salts, potassium salts, sodium salts,phosphates, and other salts of manganese, zinc, iron, copper,molybdenum, and cobalt. If necessary, amino acids and vitamins can beappropriately used in combination.

The microorganisms used in the present invention are cultured underaerobic conditions at temperatures, usually, in the range of 4-40° C.,preferably, 20-37° C.; and at pHs of 4-10, preferably, pHs of 5-9. Thecultivation time used in the present invention is set to a time orlonger than that required for the growth initiation of themicroorganisms, preferably, 10-150 hours. The concentration of dissolvedoxygen (DO) in nutrient culture media is not specifically restricted,but usually it is in the range of 0.5-20 ppm and it can be kept withinthe range by means of controlling the level of aeration, stirring,adding oxygen to air, and increasing the inner pressure of fermentors.The cultivation is freely carried out batchwise or in continuous manner.

After completion of the culture of microorganisms, the enzyme of thepresent invention is collected. Inasmuch as the activity of the enzymeis found in both cells and cell-free cultures, the latter can becollected as crude enzyme solutions and the intact cultures can be usedas crude enzyme solutions. Conventional liquid-solid separation methodscan be employed to remove cells from the cultures; methods to centrifugethe cultures, filtrate the cultures with precoat filters, and filterwith plane filters or follow fibers can be appropriately used to removecells from the cultures. As described above, cell-free cultures can beused as crude enzyme solutions, however, they may be concentrated priorto use by salting out using ammonium sulfate, sedimentation usingacetone and alcohol, concentration in vacuo, and concentration usingplane membranes and hollow fibers.

Cell-free solutions and their concentrates, which contain the enzyme ofthe present invention, can be used intact or after immobilizing theenzyme using conventional methods. In this case, for example,conjugation methods using ion-exchangers, covalent bonding/adsorptionmethods using resins and membranes, and inclusion methods usinghigh-molecular weight substances can be appropriately employed.

The enzyme solutions usually contain theα-isomaltosylglucosaccharide-forming enzyme of the present invention andα-isomaltosyl-transferring enzyme. If necessary, theα-isomaltosylglucosaccharide-forming enzyme of the present invention canbe used after being separated/purified by conventional methods. As anexample, an electrophoretically homogenousα-isomaltosylglucosaccharide-forming enzyme according to the presentinvention can be obtained by salting out to concentrate the enzyme inthe cultures, dialyzing the concentrated crude enzyme, purifying thedialyzed solution by sequential chromatographies of anion-exchangecolumn chromatography using a resin of “SEPABEADS FP-DA13”, affinitychromatography using a gel of “SEPHACRYL HR S-200”, hydrophobicchromatography using a gel of “BUTYL-TOYOPEARL 650M”, and affinitychromatography using a gel of “SEPHACRYL HR S-200”.

The α-isomaltosylglucosaccharide-forming enzyme of the present inventionhas the characteristic physicochemical properties that it forms, via theα-glucosyl-transfer, a saccharide, which has a glucose polymerizationdegree of at least three and has both the α-1,6 glucosidic linkage as alinkage at the non-reducing end and the α-1,4 glucosidic linkage otherthan the above linkage, from a material saccharide which has a glucosepolymerization degree of at least two and has the α-1,4 glucosidiclinkage as a linkage at the non-reducing end, without substantiallyincreasing the reducing power of the material saccharide; it has nodextran-forming ability; and it is inhibited by EDTA(ethylenediaminetetraacetic acid). More particularly, the enzyme has thephysicochemical properties as shown in the below; and the saccharide,which has both a glucose polymerization degree of at least two and theα-1,4 glucosidic linkage as a linkage at the non-reducing end, includes,for example, one or more saccharides selected frommaltooligosaccharides, maltodextrins, amylodextrins, amyloses,amylopectins, soluble starches, gelatinized starches, and glycogens:

(1) Action

-   -   Forming a saccharide having a glucose polymerization degree of        at least three and having both the α-1,6 glucosidic linkage as a        linkage at the non-reducing end and the α-1,4 glucosidic linkage        other than the above linkage, via the α-glucosyl-transfer from a        saccharide having a glucose polymerization degree of at least        two and having the α-1,4 glucosidic linkage as a linkage at the        non-reducing end, without substantially increasing the reducing        power of the material saccharide;

(2) Molecular Weight

-   -   Having a molecular weight of about 74,000 to about 160,000        daltons when determined on SDS-PAGE;

(3) Isoelectric Point

-   -   Having an isoelectric point of about 3.8 to about 7.8 when        determined on isoelectrophoresis using ampholine;

(4) Optimum Temperature

-   -   Having an optimum temperature of about 40° C. to about 50° C.        when incubated at a pH of 6.0 for 60 min;    -   Having an optimum temperature of about 45° C. to about 55° C.        when incubated at a pH of 6.0 for 60 min in the presence of 1 mM        Ca²⁺;    -   Having an optimum temperature of 60° C. when incubated at a pH        of 8.4 for 60 min; or    -   Having an optimum temperature of 65° C. when incubated at a pH        of 8.4 for 60 min in the presence of 1 mM Ca²⁺;

(5) Optimum pH

-   -   Having an optimum pH of about 6.0 to about 8.4 when incubated at        35° C. for 60 min;

(6) Thermal Stability

-   -   Having a thermostable region at temperatures of about 45° C. or        lower when incubated at a pH of 6.0 for 60 min,    -   Having a thermostable region at temperatures of about 50° C. or        lower when incubated at a pH of 6.0 for 60 min in the presence        of 1 mM Ca²⁺,    -   Having a thermostable region at temperatures of about 55° C. or        lower when incubated at a pH of 8.0 for 60 min, and    -   Having a thermostable region at temperatures of about 60° C. or        lower when incubated at a pH of 8.0 for 60 min in the presence        of 1 mM Ca²⁺;

(7) pH Stability

-   -   Having a stable pH region at about 4.5 to about 10.0 when        incubated at 4° C. for 24 hours; and

(8) N-Terminal Amino Acid Sequence

-   -   tyrosine-valine-serine-serine-leucine-glycine-asparagine-leucine-isoleucine,        SEQ ID NO:1    -   histidine-valine-serine-alanine-leucine-glycine-asparagine-leucine-leucine,        SEQ ID NO:11    -   alanine-proline-leucine-glycine-valine-glutamine-arginine-alanine-glutamine-phenylalanine-glutamine-serine-glycine.        SEQ NO:18

The substrates usable for the α-isomaltosyl-glucosaccharide-formingenzyme of the present invention include polysaccharides having the1,4-glucosidic linkage such as starches, amylopectins, amyloses, andglycogens; and partial starch hydrolyzates such as amylodextrins,maltodextrins, and maltooligosaccharides obtainable by partiallyhydrolyzing the above polysaccharides with amylases, acids, etc. Theseglucosyl saccharides having the α-1,4 glucosidic linkage can be furthertreated with a branching enzyme (EC 2.4.1.18) such as a branching enzymefor the substrates. Examples of partial starch hydrolyzates of glucosylsaccharides treated with amylases are those which are hydrolyzed withα-amylase (EC 3.2.1.1), β-amylase (EC 3.2.1.2), maltotriose-formingenzyme (EC 3.2.1.116), maltotetraose-forming enzyme (EC 3.2.1.60),maltopentaose-forming enzyme, and maltohexaose-forming amylase (EC3.2.1.98) as disclosed in “Handbook of Amylases and Related Enzymes”,published by Pergamon Press, Tokyo, Japan (1988). In the case ofpreparing partial starch hydrolyzates, debranching enzymes such aspulullanase (EC 3.2.1.41) and isoamylase (EC 3.2.1.68) can bearbitrarily used.

The starches as the substrates include terrestrial starches from cropssuch as corns, wheats, and rices; and subterranean starches such aspotatoes, sweet potatoes, and tapioca. Preferably, these starches aregelatinized and/or liquefied into a liquid form in use. The lower thedegree of partial hydrolysis, the higher the yield ofcyclotetrasaccharide becomes, and therefore the DE is set to a level ofabout 20 or lower, preferably, about 12 or lower, and more preferably,about five or lower.

The acceptors for transferring reaction by theα-isomaltosylglucosaccharide-forming enzyme of the present inventioninclude the above substrates per se, monosaccharides such as glucose,xylose, galactose, fructose, arabinose, fucose, sorbose, andN-acetylglucosamine; oligosaccharides such as trehalose, isomaltose,isomaltotriose, cellobiose, gentibiose, lactose, and sucrose; and otherssuch as maltitol and L-ascorbic acid.

The concentration of substrates is not specifically restricted, and theenzymatic reaction of the present invention proceeds even when used in alow concentration as low as 0.1% (w/w) (throughout the specification, “%(w/w)” is abbreviated as “%”, hereinafter, unless specified otherwise).However, one percent or higher concentrations are preferably used for anindustrial scale production. The substrate solutions may be those in asuspension form which contain incompletely-dissolved insolublesubstrates. The substrate concentration is preferably 40% or lower, andmore preferably, 30% or lower.

The temperatures for the enzymatic reaction used in the presentinvention are those which proceed the enzymatic reaction, i.e., those upto about 65° C., preferably, about 30° C. to about 55° C. The pHs forthe enzymatic reaction are usually set to 4.5-10, preferably, about 5.5to about 9. The time for the enzymatic reaction can be appropriately setdepending on the enzymatic reaction efficiency.

By contacting the α-isomaltosylglucosaccharide formed by the aboveenzymatic reaction with α-isomaltosyl enzyme, cyclotetrasaccharide isproduced in a satisfactorily yield. The α-isomaltosyl-transferringenzyme can be allowed to act on substrates after the action and theinactivation of the α-isomaltosylglucosaccharide-forming enzyme of thepresent invention. Preferably, theseα-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme can be used in combination tofacilitate the production of cyclotetrasaccharide from starches orpartial hydrolyzates thereof in a high yield of about 30%, d.s.b., orhigher, and the production from glycogen in a yield of about 80%,d.s.b., or higher. The formation mechanism of cyclotetrasaccharide bythe above combination use can be estimated as follows based on thereaction properties of the two enzymes:

-   -   (1) The α-isomaltosylglucosaccharide-forming enzyme of the        present invention acts on the α-1,4 glucosyl residue at the        non-reducing end of a saccharide, which has a glucose        polymerization of at least two and has the α-1,4 glucosidic        linkage as a linkage at the non-reducing end, such as starches,        glycogen, and partial starch hydrolyzates thereof, to release a        glucose residue; and then intermolecularly transfers the        released glucose residue to the hydroxyl group at C-6 of the        glucose of other saccharide and forms a saccharide having an        α-isomaltosyl residue at the non-reducing end;    -   (2) The α-isomaltosyl-transferring enzyme acts on the saccharide        having an α-isomaltosyl residue at the non-reducing end, and        then intermolecularly transfers the residue to the hydroxyl        group at C-3 of a glucose residue of other saccharide having an        α-isomaltosyl residue at the non-reducing end and forms a        saccharide having an isomaltosyl-1,3-isomaltosyl residue at the        non-reducing end;    -   (3) The α-isomaltosyl-transferring enzyme acts on the saccharide        having an isomaltosyl-1,3-isomaltosyl residue at the        non-reducing end to release the isomaltosyl-1,3-isomaltosyl        residue from the saccharide by the intermolecular transferring        action, and then cyclizes the released        isomaltosyl-1,3-isomaltosyl saccharide into        cyclotetrasaccharide; and    -   (4) Through the steps (1) to (3), cyclotetrasaccharide is formed        from the resulting saccharide with no        isomaltosyl-1,3-isomaltosyl residue, and the yield of        cyclotetrasaccharide is highly increased by sequentially        repeating the steps (1) to (3).

As explained above, it can be estimated that, when used in combination,the α-isomaltosylglucosaccharide-forming enzyme of the present inventionand α-isomaltosyl-transferring enzyme repeatedly act on their substratesto increase the yield of cyclotetrasaccharide.

During the cyclotetrasaccharide-forming reaction, optionally, othersaccharide-transferring enzyme(s) can be advantageously used incombination to improve the yield of cyclotetrasaccharide; when two typesof enzymes, i.e., α-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme are allowed to act, for example, on anabout 15% solution of partial starch hydrolyzate, cyclotetrasaccharideis produced in a yield of about 55%, while the use of three types ofenzymes, i.e., α-isomaltosylglucosaccharide-forming enzyme,α-isomaltosyl-transferring enzyme, and cyclomaltodextringlucanotransferase, under the same conditions as above, increases themaximum yield of cyclotetrasaccharide by about 5-10% to an improvedyield of about 60-65%.

In the case of forming cyclomaltodextrin, it can be produced by culturemethods using microorganisms capable of forming bothα-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme which specifically hydrolyzes thelinkage between the α-isomaltosyl moiety and the restingglucosylsaccharide moiety of α-isomaltosylglucosaccharide formed by theα-isomaltosylglucosaccharide-forming enzyme, and then transfers thereleased α-isomaltosyl moiety to an acceptor.

As the culture media used in the above methods using microorganisms, anysynthetic or natural media can be used as long as they containsaccharides having a glucose polymerization degree of at least two andhaving the α-1,4 glucosidic linkage as a linkage at the non-reducing endand in which the microorganisms can grow. As for the other conditionsfor culturing microorganisms, those which are used to form theα-isomaltosylglucosaccharide-forming enzyme of the present invention canbe employed.

The cultures, obtained by the above enzymatic reaction and culture, canbe used intact as solutions comprising cyclotetrasaccharide orsaccharide compositions of the same. In general, they can be purifiedbefore use in such a manner of using one or more of the followingpurification methods alone or in combination: Decoloration withactivated charcoal, desalting by ion-exchange resins in a H or OH form,and column chromatographies such as ion-exchange column chromatography,column chromatography using activated charcoal, and silica gel columnchromatography, separation using organic solvents such as alcohols andacetone, membrane separation using adequate separability, hydrolysis ofthe remaining saccharides using enzymes such as amylases includingα-amylase, β-amylase, glucoamylase (EC 3.2.1.3), and α-glucosidase (EC3.2.1.20), and hydrolysis and removal of the remaining saccharides byfermentation with yeasts or by alkaline treatment.

Particularly, ion-exchange column chromatography is preferably used asan industrial scale production method; column chromatography usingstrong-acid cation exchange resins as disclosed, for example, inJapanese Patent Kokai Nos. 23,799/83 and 72,598/83. Using the columnchromatography, the contaminating saccharides can be removed toadvantageously produce cyclotetrasaccharide with an improved content ofthe objective saccharide or saccharide compositions comprising the same.In this case, any one of fixed-bed, moving bed, and semi-moving bedmethods can be appropriately used.

The resulting cyclotetrasaccharide and saccharide compositionscomprising the same can be appropriately concentrated into syrupyproducts, and optionally they can be further dried into powderyproducts.

To produce cyclotetrasaccharide crystals, for example, highcyclotetrasaccharide content solutions, having a concentration of about30-90% and a purity of about at least 50% of cyclotetrasaccharide, areplaced in a crystallizer optionally in the presence of an organicsolvent, and then gradually cooled while stirring in the presence of0.1-20%, d.s.b., of a seed crystal to the cyclotetrasaccharide attemperatures of 95° C. or lower, preferably, 10-90° C., to obtainmassecuites. The methods to collect cyclotetrasaccharide crystals andmolasses with such crystals include, for example, conventional methodssuch as separation, block pulverization, fluidized granulation, andspray drying methods.

The resulting cyclotetrasaccharide according to the present invention isa stable, high-quality, low sweetness, non-reducing white power, and isalmost free of browning, smelling, and deterioration of materials evenwhen mixed or processed therewith: The materials are particularly, forexample, amino acid-containing substances such as amino acids,oligopeptides, and proteins.

Since cyclotetrasaccharide has an inclusion ability, it effectivelyinhibits the dispersion and quality deterioration of flavorfulcomponents and effective ingredients, and stably retains them. For sucha purpose, the combination use of cyclotetrasaccharide and other cyclicsaccharide(s) such as cyclodextrins, branched cyclodextrins,cyclodextrans, and cyclofructans can be advantageously used to improvethe level of the inclusion ability of cyclotetrasaccharide, ifnecessary. The above cyclic saccharides such as cyclodextrins usable inthe present invention should not be restricted to those with a highpurity, and can be advantageously a relatively-low purity ofcyclotetrasaccharide such as partial starch hydrolyzates containing alarge quantity of maltodextrins and cyclodextrins.

Since cyclotetrasaccharide is not hydrolyzed by amylase andα-glucosidase, it is substantially free of assimilation by the body whenorally administered. Also, the saccharide is not substantiallyassimilated by intestinal microorganisms, and therefore it can be usedas an extremely-low caloric water-soluble dietary fiber.Cyclotetrasaccharide can be also used as a sweetener substantially freefrom causing dental caries because it is scarcely assimilated by dentalcaries-inducing microorganisms. The saccharide prevents the adhesion andsolidification of powdery products. The cyclotetrasaccharide of thepresent invention per se is a natural sweetener with a satisfactorystability but with no toxicity, harm, and side effect, and because ofthese it can be advantageously used for tablets and sugar-coated tabletsin combination with binders such as pullulan, hydroxyethyl starch, andpolyvinylpyrrolidone. Furthermore, cyclotetrasaccharide has propertiesof osmosis-controlling ability, filler-imparting ability,gloss-imparting ability, moisture-retaining ability, viscosity,crystallization prevention ability for other saccharides, insubstantialfermentability, etc.

Thus, the cyclotetrasaccharide and the saccharide compositionscomprising the same of the present invention can be arbitrary used as asweetener, taste-improving agent, quality-improving agent, stabilizer,preventive of discoloration, excipient, etc., in a variety ofcompositions such as food products, tobaccos, cigarettes, feeds, petfoods, cosmetics, and pharmaceuticals.

The cyclotetrasaccharide and the saccharide compositions comprising thesame of the present invention can be used in combination with one ormore other sweeteners, for example, powdered syrup, glucose, isomerizedsugar, sucrose, maltose, trehalose, honey, maple sugar, sorbitol,maltitol, dihydrochalcone, stevioside, α-glycosyl stevioside, sweetenerof Momordica grosvenori, glycyrrhizin, thaumatin, L-aspartylL-phenylalanine methyl ester, saccharin, acesulfame K, sucralose,glycine, and alanine; and fillers such as dextrins, starches, andlactose. Particularly, the cyclotetrasaccharide and the saccharidecompositions comprising the same can be suitably used as a low-caloricsweetener, diet sweetener, or the like in combination with one or morelow-caloric sweeteners such as meso-erythritol, xylitol, and maltitol;and/or one or more sweeteners with a relatively-high sweetening powersuch as α-glycosyl stevioside, thaumatin, L-aspartyl L-phenylalaninemethyl ester, saccharin, acesulfame K, and sucralose.

The cyclotetrasaccharide and the saccharide compositions comprising thesame of the present invention can be arbitrarily used intact or aftermixing with fillers, excipients, binders, etc., and then formed intoproducts with different shapes such as granules, spheres, plates, cubes,and tablets.

The cyclotetrasaccharide and the saccharide compositions comprising thesame of the present invention well harmonize with other tastablematerials having sour-, acid-, salty-, delicious-, astringent-, andbitter-tastes; and have a satisfactorily high acid- and heat-tolerance.Thus, they can be favorably used as sweeteners, taste-improving agents,quality-improving agents, etc., to sweeten and/or improve the taste andquality of food products in general, for example, a soy sauce, powderedsoy sauce, miso, “funmatsu-miso” (a powdered miso), “moromi” (a refinedsake), “hishio” (a refined soy sauce), “furikake” (a seasoned fishmeal), mayonnaise, dressing, vinegar, “sanbai-zu” (a sauce of sugar, soysauce and vinegar), “funmatsu-sushi-su” (powdered vinegar for sushi),“chuka-no-moto” (an instant mix for Chinese dish), “tentsuyu” (a saucefor Japanese deep-fat fried food), “mentsuyu” (a sauce for Japanesevermicelli), sauce, catsup, “yakiniku-no-tare” (a sauce for Japanesegrilled meat), curry roux, instant stew mix, instant soup mix,“dashi-no-moto” (an instant stock mix), mixed seasoning, “mirin” (asweet sake), “shin-mirin” (a synthetic mirin), table sugar, and coffeesugar. Also, the cyclotetrasaccharide and the saccharide compositionscomprising the same of the present invention can be arbitrarily used tosweeten and improve the taste and quality of “wagashi” (Japanese cakes)such as “senbei” (a rice cracker), “arare” (a rice cake cube), “okoshi”(a millet-and-rice cake), “gyuhi” (a starch paste), “mochi” (a ricepaste) and the like, “manju” (a bun with a bean-jam), “uiro” (a sweetrice jelly), “an” (a bean jam) and the like, “yokan” (a sweet jelly ofbeans), “mizu-yokan” (a soft adzuki-bean jelly), “kingyoku” (a kind ofyokan), jelly, pao de Castella, and “amedama” (a Japanese toffee);Western confectioneries such as a bun, biscuit, cracker, cookie, pie,pudding, butter cream, custard cream, cream puff, waffle, sponge cake,doughnut, chocolate, chewing gum, caramel, nougat, and candy; frozendesserts such as an ice cream and sherbet; syrups such as a“kajitsu-no-syrup-zuke” (a preserved fruit) and “korimitsu” (a sugarsyrup for shaved ice); pastes such as a flour paste, peanut paste, andfruit paste; processed fruits and vegetables such as a jam, marmalade,“syrup-zuke” (fruit pickles), and “toka” (conserves); pickles andpickled products such as a “fukujin-zuke” (red colored radish pickles),“bettara-zuke” (a kind of whole fresh radish pickles), “senmai-zuke” (akind of sliced fresh radish pickles), and “rakkyo-zuke” (pickledshallots); premixes for pickles and pickled products such as a“takuan-zuke-no-moto” (a premix for pickled radish), and“hakusai-zuke-no-moto” (a premix for fresh white rape pickles); meatproducts such as a ham and sausage; products of fish meat such as a fishham, fish sausage, “kamaboko” (a steamed fish paste), “chikuwa” (a kindof fish paste), and “tenpura” (a Japanese deep-fat fried fish paste);“chinmi” (relish) such as a “uni-no-shiokara” (salted guts of seaurchin), “ika-no-shiokara” (salted guts of squid), “su-konbu” (processedtangle), “saki-surume” (dried squid strips), “fugu-no-mirin-boshi” (adried mirin-seasoned swellfish), seasoned fish flour such as of Pacificcod, sea bream, shrimp, etc; “tsukudani” (foods boiled down in soysauce) such as those of laver, edible wild plants, dried squid, smallfish, and shellfish; daily dishes such as a “nimame” (cooked beans),potato salad, and “konbu-maki” (a tangle roll); milk products; cannedand bottled products such as those of meat, fish meat, fruit, andvegetable; alcoholic beverages such as a synthetic sake, fermentedliquor, sake, fruit wine, sparkling alcoholic beverage, beer; softdrinks such as a coffee, cocoa, juice, carbonated beverage, sour milkbeverage, and beverage containing a lactic acid bacterium; instant foodproducts such as instant pudding mix, instant hot cake mix, instantjuice or soft drink, instant coffee, “sokuseki-shiruko” (an instant mixof adzuki-bean soup with rice cake), and instant soup mix; and otherfoods and beverages such as solid foods for babies, foods for therapy,health/tonic drinks, peptide foods, and frozen foods. Thecyclotetrasaccharide and the saccharide compositions comprising the sameof the present invention can be arbitrary used to prolong or retain theflavor and taste of fresh-baked Japanese and Western confectioneries andto improve the taste preference of feeds and pet foods for animals andpets such as domestic animals, poultry, honey bees, silk worms, andfish; and also they can be arbitrary arbitrarily used as a sweetener,taste-improving agent, flavoring substance, quality-improving agent, andstabilizer in other products in a paste or liquid form such as atobacco, cigarette, tooth paste, lipstick, rouge, lip cream, internalliquid medicine, tablet, troche, cod liver oil in the form of drop,cachou, oral refrigerant, gargle, cosmetic, and pharmaceutical. Whenused as a quality-improving agent or stabilizer, thecyclotetrasaccharide and the saccharide compositions comprising the sameof the present invention can be arbitrarily used in biologically activesubstances susceptible to lose their effective ingredients andactivities, as well as in health foods and pharmaceuticals containingthe biologically active substances. Examples of such biologically activesubstances are liquid preparations containing lymphokines such as α-, β-and γ-interferons, tumor necrosis factor-α (TNF-α), tumor necrosisfactor-β (TNF-β), macrophage migration inhibitory factor,colony-stimulating factor, transfer factor, and interleukin 2; liquidpreparations containing hormones such as insulin, growth hormone,prolactin, erythropoietin, and follicle-stimulating hormone; biologicalpreparations such as BCG vaccine, Japanese encephalitis vaccine, measlesvaccine, live polio vaccine, smallpox vaccine, tetanus toxoid,Trimeresurus antitoxin, and human immunoglobulin; antibiotics such aspenicillin, erythromycin, chloramphenicol, tetracycline, streptomycin,and kanamycin sulfate; liquid preparations containing vitamins such asthiamine, riboflavin, L-ascorbic acid, cod liver oil, carotenoid,ergosterol, and tocopherol; highly unsaturated fatty acids and esterderivatives thereof such as EPA, DHA, and arachidonic acid; solutions ofenzymes such as lipase, elastase, urokinase, protease, β-amylase,isoamylase, glucanase, and lactase; extracts such as ginseng extract,snapping turtle extract, chlorella extract, aloe extract, and propolisextract; and royal jelly. By using the cyclotetrasaccharide and thesaccharide compositions comprising the same of the present invention,the above biologically active substances and other pastes of livingmicroorganisms such as viruses, lactic acid bacteria, and yeasts can bearbitrarily prepared into health foods and pharmaceuticals in a liquid,paste, or solid form, which have a satisfactorily-high stability andquality with less fear of losing or inactivating their effectiveingredients and activities.

As mentioned above, the following effects and features are alsoeffectively exerted when used with other ingredients which are generallyused externally: The effects of preventing the volatilization or thekeeping of ingredients of fragrances and fragrances, preventingsyneresis, crystallization of other saccharides, and deterioration ofproteins, lipids, and active ingredients, retaining moisture, andstabilizing emulsified conditions, which are exerted by thecyclotetrasaccharide and the saccharide compositions comprising thesame; and the features of stability and filler-imparting abilityinherent to the cyclotetrasaccharide and the saccharides.

Similarly as other naturally occurring saccharides, since thecyclotetrasaccharide and the saccharide compositions comprising the sameof the present invention quite scarcely stimulate the skin when appliedthereupon and effectively retain the moisture in the skin, they can beadvantageously incorporated into external dermal compositions for use.In the external dermal compositions, the cyclotetrasaccharide and thesaccharide compositions comprising the same of the present invention canbe usually used in an appropriate combination with one or moredermatologically applicable other ingredients of oils and lipids, waxes,hydrocarbons, fatty acids, esters, alcohols, surfactants, dyes,fragrances, hormones, vitamins, plant extracts, animal extracts,microbial extracts, salts, ultraviolet absorbents, photosensitizingdyes, antioxidants, antiseptics/bactericides,antiperspirants/deodorants, refreshments, chelating agents, skinwhitening agents, anti-inflammatories, enzymes, saccharides, aminoacids, and thickening agents. For example, in the field of cosmetics,the external dermal compositions can be provided in the form of alotion, cream, milky lotion, gel, powder, paste, or block, for example,cleaning cosmetics such as soaps, cosmetic soaps, washing powders forthe skin, face washing creams, facial rinses, body shampoos, bodyrinses, shampoos, and powders for washing hair; cosmetics for hair suchas set lotions, hair blows, stick pomades, hair creams, pomades, hairsprays, hair liquids, hair tonics, hair lotions, hair restorers, hairdyes, treatments for scalp, hair cosmetics, gloss-imparting hair oils,hair oils, and combing oils; base cosmetics such as cosmetic lotions,vanishing creams, emollient creams, emollient lotions, cosmetic packs inthe form of a jelly peel off, jelly wiping, paste washing, powders,cleansing creams, cold creams, hand creams, hand lotions, milky lotions,moisture-imparting liquids, after/before shaving lotions, after shavingcreams, after shaving foams, before shaving creams, and baby oils;makeup cosmetics such as foundations in the form of a liquid, cream orsolid, talcum powders, baby powders, body powders, perfume powders,makeup bases, powders in the form of a cream, paste, liquid, solid orpowder, eye shadows, eye creams, mascaras, eyebrow pencils, eyelashmakeups, rouges, rouge lotions; perfume cosmetics such as perfumes,paste/powder perfumes, eau de Colognes, perfume Colognes, and eau detoilette; suntan and suntan preventive cosmetics such as suntan creams,suntan lotions, and suntan oils; nail cosmetics such as manicures,pedicures, nail colors, nail lacquers, and nail makeup materials;eyeliner cosmetics; rouges and lipsticks such as lipsticks, lipcreams,paste rouges, and lip-glosses; oral cosmetics such as tooth pastes andmouth washes; and bath cosmetics such as bath salts/oils, and bathcosmetic materials. In the field of pharmaceuticals, the external dermalcompositions can be provided in the form of a wet compresses, sprays,applications, bath agents, sticking agents, ointments, pastes,embrocations, lotions, and cataplasms.

Concrete examples of the other ingredients, which can be incorporatedinto the external dermal compositions along with thecyclotetrasaccharide and the saccharide compositions comprising thesame, are oils and fats including plant oils in the form of a liquid atambient temperature such as an avocado oil, almond oil, olive oil,sesame oil, safflower oil, soy bean oil, camellia oil, persic oil,castor oil, and cotton seed oil; plant fats in the form of a solid atambient temperature such as a cacao fat, palm fat/oil, and vegetablewax; and animal oils such as mink oil, egg yolk oil, and turtle oil.

Examples of the waxes usable in the present invention are plant waxessuch as a hohoba oil, carnauba was, and candelilla wax; animal waxessuch as a sperm oil, Baird's beaked while oil, beeswax, whale oil, andlanolin; and mineral oils such as a montan wax.

The carbohydrates usable in the present invention are, for example,mineral carbohydrates such as a paraffin or solid paraffin, liquidparaffin, ceresin, microcrystalline wax, and petrolatum; and animalhydrocarbons such as squalane and squalene.

Examples of the fatty acids usable in the present invention are lauricacid, myristic acid, palmitic acid, stearic acid, oleic acid, behenicacid, undecylenic acid, lanolin fatty acid, hard lanolin fatty acid,soft lanolin fatty acid, isostearic acid, and derivatives thereof.

The alcohols usable in the present invention are, for example, higheralcohols including polyalcohols such as lauryl alcohol, cetanol,setostearyl alcohol, stearyl alcohol, oleyl alcohol, behenyl alcohol,lanolin alcohol, hydrogenated lanolin alcohol, hexyldecanol,octyldodecanol, and polyethylene glycol; lower alcohols includingpolyalcohols such as ethanol, propanol, isopropanol, butanol, ethyleneglycol, propylene glycol, and glycerine; and derivatives thereof.

Examples of the esters usable in the present invention are hexyllaurate, isopropyl myristate, myristyl myristate, cetyl myristate, octyldodecyl myristate, isopropyl palmitate, butyl stearate, cholesterylstearate, cholesteryl acetate, cholesteryl n-lactate, cholesterylcaproate, cholesteryl laurate, cholesteryl myristate, cholesterylpalmitate, cholesteryl stearate, cholesteryl 12-hydroxystearate, decyloleate, octyldodecyl oleate, isopropyl lanolin fatty acid, glycerinetrimyristate, propylene glycol dioleate, myristyl lactate, cetyllactate, lanolin acetate, hexyldecyl dimethyloctanoate, and derivativesthereof.

The surfactants usable in the present invention are, for example,anionic surfactants such as zinc laurate, zinc myristate, zincpalmitate, magnesium stearate, sodium lauryl sulfate, sodiumpolyoxyethylene laurylether sulfate, triethanolamine polyoxyethylenelaurylether sulfate, polyoxyethylene cetylether phosphate,polyoxyethylene alkylphenylether phosphate, sodium N-lauroylsarcosinate, coconut fatty acid sarcosinate triethanolamine, coconutfatty acid sodium methyltaurate, and soybean phospholipid; cationicsurfactants such as stearyltrimethylammonium chloride,distearyldimethylammonium chloride, benzalkonium chloride,cetylpyridinium chloride, alkylisoquinolinium bromide, anddodecyldimethyl 2-phenoxyethylammonium bromide; amphoteric ionsurfactants such as sodium β-laurylaminopropionate, betainelauryldimethylamino acetate, and 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine; non-ionic surfactants such as glycerylmonostearate, self-emulsifying, glyceryl monostearate, lipophilic,sorbitan monolaurate, sorbitan monooleate, sucrose fatty acid ester,undecylenic acid monoethanolamide, coconut oil diethanolamide,polyethylene glycol monooleate, myristyl lactate, cetyl lactate,polyoxyethylene cetylether, polyoxyethylene octylphenylether,polyoxyethylene sorbitol monolaurate, polyoxyethylene sorbitanmonolaurate, polyoxyethylene sorbitan monostearate, polyoxyethylenesorbitan trioleate, polyoxyethylene sorbitol tetraoleate,polyoxyethylene castor oil, and polyoxyethylene hydrogenated castor oil;and derivatives thereof.

Examples of the dyes usable in the present invention are red tar dyessuch as amaranth, erythrosine, rose bengal, acid red, lake red C, litholred, rhodamine, brilliant lake red, eosine YS, violamine R, brilliantfast scarlet, Ponceau R, orange tar dyes such as dibromofluorescein,permanent orange, erythrosine yellow NA, and orange I; yellow tar dyessuch as tartrazine, sunset yellow, uranin, benzidine yellow G, naphtholyellow S, and yellow AB; green tar dyes such as fast green FCF, alizarincyanine green F, light green SF yellow, and naphthol green B; blue tardyes such as brilliant blue FCF, indigo carmine, indigo, patent blue NA,carbanthrene blue, and sudan blue; brown tar dyes such as resorcinbrown; purple tar dyes such as alizarin purple and alizarin purple;black tar dyes such as naphthol blue black; inorganic pigments such aszinc oxide, titanium oxide, cobalt hydroxide, aluminum hydroxide, talc,kaolin, mica, bentonite, manganese violet, and mica titanium; carotenoidpigments such as β-carotenoid, lycopene, and crocin; flavonoid pigmentssuch as sisonine, saffrol yellow, rutin, and quercetin; flavin pigmentssuch as riboflavin; quinone pigments such as cochineal, alizarine, andshikonin; and derivatives thereof.

The fragrances used generally in external dermal uses can be roughlyclassified into natural plant and animal fragrances, syntheticfragrances, and mixtures thereof in an appropriate combination. Examplesof the animal fragrances include musk, civetone, and ambergris. Theplant fragrances are, for example, distillations, i.e., essential oils,obtainable by distilling, for example, with water vapor anise seeds,basil leaves, caraway fruit, cinnamon barks, coriander seeds, lavenderflowers, nutmeg seeds, peppermint leaves, rose flowers, rosemaryflowers, seeds, and leaves, and thyme leaves; extracts classifiedgenerally into absolutes, resinoids, oleo resins, and tincturesdepending on properties and processes. Examples of the syntheticfragrances are acetophenone, anisole, benzyl alcohol, butyl acetate,camphor, citral, citronellol, cuminaldehyde, estragol, ethylvaniline,geranyl acetate, linarol, menthol, methyl p-cresol, methyl salicylate,phenyl acetate, vanillin, and derivatives thereof. In the presentinvention, flavor compositions mixed with the aforesaid fragrances in anappropriate combination can be arbitrarily used.

The hormones usable in the present invention include, for example,follicle hormones such as estrone and estradiol; gestagens such asprogesterone and pregnenolone; and adrenal cortex hormones such ascortisone, hydrocortisone, and prednisolone. The vitamins usable in thepresent invention are, for example, vitamin A compounds such as retinol,retinoic acid, α-, β- and γ-carotenes, and derivatives thereof; vitaminB compounds such as thiamine (vitamin B1), riboflavin (vitamin B2),vitamin B6 including pyridoxine, pyridoxal, and pyridoxamine, andderivatives thereof; vitamin C compounds such as L-ascorbic acid,2-O-α-D-glucosyl-L-ascorbic acid, acyl derivatives, alias lipophilicvitamin C, of L-ascorbic acid and glycosyl-L-ascorbic acid, and otherL-ascorbic acid derivatives such as L-ascorbic acid sulfate ester;vitamin D compounds such as ergocalciferol, cholecalciferol, andderivatives thereof; and vitamin E compounds such as α-, β-, γ- andδ-tocopherol, α-, β-, γ- and δ-tocotrienol, and derivatives thereof.

Examples of the plant extracts usable in the present invention are, inaddition to the aforesaid plant extracts used as fragrances, extractssuch as those of chamomile, sage, aloe, scarlet sage, Angelica keiskei,avocado, nettle, fennel, oolong tea, coak tree bark, barley, Abelmoschusesculentus, allspice, seaweed, chinese quince, licorice, quince seed,gardenia, Sasa albo-marginata, cinnamon, black tea, rice bran, fermentedrice bran, Stevia rebaudiana, celery, Japanese green gentian, soy bean,thyme, tea, common camellia, Ligusticum acutilobum, corn, carrot, Rosarugosa, hinoki (Japanese cypress), dishcloth gourd, safflower, pine,peach, eucalyptus, creeping saxifrage, yuzu (citron), lily, Job's tears,Mugwort, Cyanophta (blue-green algae), seaweed, apple, Serratiamarcescens, and lettuce; and compounds isolated from plants such ashinokitiol, azulene, chlorophyll, and glycyrrhizin. The animal extractsusable in the present invention include placenta extracts.

Examples of the extracts of microorganisms are yeast extracts. The saltsusable in the external dermal composition of the present inventionadvantageously include those which can be used generally in conventionalexternal dermal compositions, as well as sea water, deep sea water,dried ingredients of sea water, and natural salts, including those inthe form of a liquid, such as mineral salts.

The ultraviolet absorbers usable in the present invention include, forexample, p-aminobenzoic acid, p-dimethylaminobenzoic acidethylhexylester, p-methoxycinnamic acid ethylhexylester,2-(hydroxy-5-methylphenyl)benzotriazole, oxibenzozone, urocanic acid,ethyl urocanate, and derivatives thereof; organic substances capable ofshielding ultraviolet rays such as 5-chlorouracil, and guanine cytosine.Examples of the photosensitive dyes usable in the present invention are2,2′[3′-[2-(3-heptyl-4-methyl-2-thiazolin-2-ylidene)ethyridene]propenylene]bis[3-heptyl-4-methylthiazolinium iodide] alias “PLATONIN”,2-[2-(3-heptyl-4-methyl-2-thiazolin-2-ylidene)methine]-3-heptyl-4-methylthiazolinium iodide alias “PIONIN”,6-[2-[(5-bromo-2-pyridyl)amino]vinyl]-1-ethyl-2-picolinium iodide alas“TAKANAL”, 2-(2-anilino vinyl)-3,4-dimethyl-oxazolinium iodide alas“LUMINEX”, and derivatives thereof.

In addition to the aforesaid compounds having anti-oxidation ability,the antioxidants usable in the present invention include, for example,propyl gallate, butyl gallate, octyl gallate, dodecyl gallate,nordihydroguaiaretic acid (NDGA), t-butylhydroxyanisole (BHA), butylatedhydroxytoluene (BHT), 4-hydroxymethyl-1-2,6-di-t-butylphenol, andderivatives thereof.

Examples of the aseptics and bactericides usable in the presentinvention include, in addition to the aforesaid compounds with asepticor bactericidal activities, phenol compounds such as phenol, p-chlorometacresol, resorcin, p-oxy benzoate, and cresol; acid compoundsincluding those in a salt form such as benzoic acid, sorbic acid,salicylic acid, and boric acid; bisphenol halides such ashexachlorophene, bithionol, and dichlorophene; amides such as3,4,4′-trichlorocarvaniride, undecylenic acid monoethanolamide;quaternary ammonium compounds such as benzalkonium chloride,benzethonium chloride, and decalinium chloride; chlorhexidinehydrochloride, 1-hydroxypyridine-2-thione, lysozyme chloride; andderivatives thereof.

The antiperspirants/deodorants usable in the present invention are, forexample, aluminum chloride, zinc chloride, chlorohydroxy aluminum,aluminum chlorohydroxy allantoinate, aluminum dihydroxy allantoinate,and aluminum chlorohydrate. Examples of the refreshments usable in thepresent invention include menthol, mint/peppermint oil, camphor, thymol,spirantol, and methyl salicylic acid. The chelating agents usable in thepresent invention are, for example, derivatives ofethylenediaminetetraacetic acid, tripolyphosphoric acid, hexamethacrylicacid, dihydroethylglycine, citric acid, tartaric acid, gluconic acid,and sugar acid.

In addition to the aforesaid compounds with skin whitening activity, theskin whitening agents usable in the present invention are, for example,nucleic acids such as antisense oligonucleotides including antisenseoligonucleotides to a tyrosinase gene; kojic acid, lactic acid,anthranilic acid, cumarin, benzotriazole, imidazoline, pyrimidine,dioxane, furan, pyrone, nicotinic acid, arbutin, baicalin, baicalein,and berberine, and derivatives thereof; melanin formation inhibitors,tyrosinase formation inhibitors, and tyrosinase inhibitors.

Examples of the anti-inflammatory agents usable in the present inventioninclude, in addition to the aforesaid those with such anti-inflammatoryactivity, for example, allantoin, allantoin acetyl-DL-methionine,β-glycyrrhetinic acid allantoinate, ichthammol, indomethacin,acetylsalicylic acid, diphenhydramine chloride, guaiazulene, camazulene,chlorpheniramine maleate, glycyrrhizinic acid, glycyrrhetinic acid, andoriental gromurel extract. Examples of the enzymes usable in the presentinvention are those from microorganisms of the genera Bacillus andStreptomyces, and yeasts; and those from plants and animals such asprotease, lipase, and lysozyme.

The saccharides usable in the present invention are, for example,oligosaccharides such as sucrose, maltose, fructose, lactose, andtrehalose; cyclic saccharides, excluding cyclotetrasaccharide, such ascyclodextrins; sugar alcohols such as maltitol, sorbitol, mannitol,xylitol, and arabitol; polysaccharides such as hyaluronic acid,chondroitin sulfate, pullulan, cellulose, starch, dextran, pectin,carrageenan, guar gum, corn syrup, gum arabic, tragacanth gum, xanthangum, and chitin, their derivative and partial hydrolyzates. Examples ofthe amino acids usable in the present invention are glycine, serine,threonine, tyrosine, cysteine, cystine, asparagine, glutamine,2-pyrrolidone-5-carboxylic acid, hydroxyproline, pipecolic acid,sarcosine, homocysteine, homoserine, citrulline, aspartic acid, glutamicacid, cysteine sulfonic acid, argininosuccinic acid, arginine, lysine,histidine, ornithine, alanine, valine, leucine, isoleucine, methionine,phenylalanine, tryptophane, proline, β-alanine, taurine, β-aminobutyricacid, γ-aminobutyric acid, and salts thereof.

The thickening agents usable in the present invention includes, inaddition to the aforesaid compounds having viscosity-imparting ability,for example, water-soluble high molecular substances such as quinceseed, sodium alginate, cationated cellulose, hydroxyethyl cellulose,carboxymethyl cellulose, carboxymethyl starch, propylene glycolalginate, collagen, keratin, hydroxypropyl trimethylammonium chlorideether, poly vinyl alcohol, polyvinylpyrrolidone,polyvinylpyrrolidone-vinylacetate copolymer, polyethylene imine, sodiumpolyacrylate, polyvinylmethyl ether, and carboxyvinylpolymer;electrolytes such as sodium chloride, potassium chloride, and sodiumsulfate; and oily materials.

Although the above examples may not completely cover all the compatiblesalts of the above-exemplified compounds/ingredients if they have suchsalts, any salt acceptable for external dermal agents other than theabove-exemplified salts can be arbitrarily used in the presentinvention.

The methods for incorporating the cyclotetrasaccharide or the saccharidecompositions comprising the same according to the present invention intothe aforesaid compositions are those which can incorporate thecyclotetrasaccharide and the saccharide compositions into a variety ofcompositions before completion of their processings, and which can beappropriately selected among the following conventional methods; mixing,kneading, dissolving, melting, soaking, penetrating, dispersing,applying, coating, spraying, injecting, crystallizing, and solidifying.The amount of the cyclotetrasaccharide or the saccharide compositionscomprising the same to be preferably incorporated into the finalcompositions is usually in an amount of at least 0.1%, desirably, atleast 1%.

The following experiments explain the present invention in detail:

EXPERIMENT 1 Preparation of Non-Reducing Cyclotetrasaccharide byCulturing

A liquid medium consisting of 5% (w/v) of “PINE-DEX #1”, a partialstarch hydrolysate commercialized by Matsutani Chemical Ind., Tokyo,Japan, 1.5% (w/v) of “ASAHIMEAST”, a yeast extract commercialized byAsahi Breweries, Ltd., Tokyo, Japan, 0.1% (w/v) of dipotassiumphosphate, 0.06% (w/v) of sodium phosphate dodecahydrate, 0.05% (w/v)magnesium sulfate heptahydrate, and water was placed in a 500-mlErlenmeyer flask in an amount of 100 ml, sterilized by autoclaving at121° C. for 20 min, cooled, and then seeded with Bacillus globisporus C9strain, FERM BP-7143, followed by culturing under rotary-shakingconditions at 27° C. and 230 rpm for 48 hours and centrifuging theresulting culture to remove cells to obtain a supernatant. Thesupernatant was autoclaved at 120° C. for 15 min and then cooled, andthe resulting insoluble substances were removed by centrifugation toobtain a supernatant.

To examine the saccharides in the supernatant, they were separated fromthe supernatant by silica gel thin-layer chromatography (abbreviated as“TLC” hereinafter) using, as a developer, a mixture solution ofn-butanol, pyridine, and water (=6:4:1), and, as a thin-layer plate,“KIESELGEL 60”, an aluminum plate (20×20 cm) for TLC commercialized byMerck & Co., Inc., Rahway, USA.

The coloration of the separated total sugars by the sulfuricacid-methanol method and the reducing saccharides by thediphenylamine-aniline method detected that a non-reducing saccharide waspositive on the former detection method but negative on the latterdetection method, and had an Rf value of 0.31.

About 90 ml of the supernatant before the saccharide detection wasadjusted to pH 5.0 and 45° C. and then incubated for 24 hours afteradmixed with 1,500 units per gram of solids of “TRANSGLUCOSIDASE LAMANO™”, an α-glucosidase commercialized by Amano Pharmaceutical Co.,Ltd., Aichi, Japan, and 75 units per gram of solids of a glucoamylasecommercialized by Nagase Biochemicals, Ltd., Kyoto, Japan. Thereafter,the resulting culture was adjusted to pH 12 by the addition of sodiumhydroxide and boiled for two hours to decompose the remaining reducingsugars. After removing insoluble substances by filtration, the resultingsolution was decolored and desalted with “DIAION PK218” and “DIAIONWA30”, cation exchange resins commercialized by Mitsubishi ChemicalIndustries, Ltd., Tokyo, Japan, and further desalted with “DIAIONSK-1B”, commercialized by Mitsubishi Chemical Industries, Ltd., Tokyo,Japan, and “AMBERLITE IRA411”, an anion exchange resin commercialized byJapan Organo Co., Ltd., Tokyo, Japan, followed by decoloring with anactivated charcoal, membrane filtered, concentrated by an evaporator,and lyophilized in vacuo to obtain about 0.6 g, d.s.b., of a saccharidepowder.

The analysis of the saccharide on high-performance liquid chromatography(abbreviated as “HPLC” hereinafter) detected a single peak at an elutiontime of 10.84 min as shown in FIG. 1, and revealed that the saccharidehad a high purity of 99.9% or higher. HPLC was carried out using“SHOWDEX KS-801 column”, Showa Denko K.K., Tokyo, Japan, at a columntemperature of 60° C. and a flow rate of 0.5 ml/min of water, and using“RI-8012”, a differential refractometer commercialized by TosohCorporation, Tokyo, Japan.

When measured for reducing power of the saccharide on theSomogyi-Nelson's method, the reducing power was below a detectablelevel, revealing that the specimen was substantially a non-reducingsaccharide.

EXPERIMENT 2 Structure Analysis on Non-Reducing Saccharide

Fast atom bombardment mass spectrometry (called “FAB-MS”) of anon-reducing saccharide, obtained by the method in Experiment 1,significantly detected a proton-addition-molecular ion with a massnumber of 649, and this meant that the saccharide had a mass number of648.

According to conventional manner, the saccharide was hydrolyzed withsulfuric acid and then analyzed for sugar composition. As a result, onlyD-glucose was detected, revealing that the saccharide was composed ofD-glucose molecules or cyclotetrasaccharide composed of four D-glucosemolecules in view of the above mass number.

Nuclear magnetic resonance analysis (called “NMR”) of the saccharidegave a ¹H-NMR spectrum as shown in FIG. 2 and a ¹³C-NMR spectrum asshown in FIG. 3, and these spectra were compared with those of knownsaccharides, revealing that they were coincided with a non-reducingcyclic saccharide,cyclo{66)-α-D-glucopyranosyl-(163)-α-D-glucopyranosyl-(166)-α-D-glucopyranosyl-(163)-α-D-glucopyranosyl-(16}as disclosed in “European Journal of Biochemistry”, pp. 641-648 (1994).The data confirmed that the saccharide of the present invention was acyclotetrasaccharide as shown in FIG. 4, i.e.,cyclo{66)-α-D-glucopyranosyl-(163)-α-D-glucopyranosyl-(166)-α-D-glucopyranosyl-(163)-α-D-glucopyranosyl-(16}.

EXPERIMENT 3 Production of α-isomaltosylglucosaccharide-forming Enzymefrom Bacillus globisporus C9 Strain C9

A liquid culture medium consisting of 4.0% (w/v) of “PINE-DEX #4”, apartial starch hydrolysate commercialized by Matsutani Chemical Ind.,Tokyo, Japan, 1.8% (w/v) of “ASAHIMEAST”, a yeast extract commercializedby Asahi Breweries, Ltd., Tokyo, Japan, 0.1% (w/v) of dipotassiumphosphate, 0.06% (w/v) of sodium phosphate dodecahydrate, 0.05% (w/v)magnesium sulfate heptahydrate, and water was placed in 500-mlErlenmeyer flasks in a respective amount of 100 ml, sterilized byautoclaving at 121° C. for 20 min, cooled, and then seeded with Bacillusglobisporus C9 strain, FERM BP-7143, followed by culturing underrotary-shaking conditions at 27° C. and 230 rpm for 48 hours for a seedculture.

About 20 L of a fresh preparation of the same liquid culture medium asused in the above seed culture were placed in a 30-L fermentor,sterilized by heating, and then cooled to 27° C. and inoculated with 1%(v/v) of the seed culture, followed by culturing at 27° C. and pH6.0-8.0 for 48 hours under aeration-agitation conditions. Aftercompletion of the culture, the resulting culture, which had about 0.45unit/ml of the α-isomaltosylglucosaccharide-forming enzyme of thepresent invention, about 1.5 units/ml of α-isomaltosyl-transferringenzyme, and about 0.95 unit/ml of cyclotetrasaccharide-forming activity,was centrifuged at 10,000 rpm for 30 min to obtain about 18 L of asupernatant. When measured for enzymatic activity, the supernatant hadabout 0.45 unit/ml of the α-isomaltosylglucosaccharide-forming enzyme ofthe present invention, i.e., a total enzymatic activity of about 8,110units; about 1.5 units/ml of α-isomaltosyl-transferring enzyme, i.e., atotal enzymatic activity of about 26,900 units; and about 0.95 unit/mlof cyclotetrasaccharide-forming activity, i.e., a total enzymaticactivity of about 17,100 units.

The activities of these enzymes were assayed as follows: Theα-isomaltosylglucosaccharide-forming enzyme of the present invention wasassayed for enzymatic activity by dissolving maltotriose in 100 mMacetate buffer (pH 6.0) to give a concentration of 2% (w/v) for asubstrate solution, adding a 0.5 ml of an enzyme solution to a 0.5 ml ofthe substrate solution, enzymatically reacting the mixture solution at35° C. for 60 min, suspending the reaction mixture by boiling for 10min, and quantifying maltose, among the isomaltosyl maltose and maltoseformed in the reaction mixture, on HPLC as disclosed in Experiment 1.One unit activity of the α-isomaltosylglucosaccharide-forming enzyme isdefined as the enzyme amount that forms one micromole of maltose perminute under the above enzymatic reaction conditions. Throughout thespecification, the enzymatic activity of theα-isomaltosylglucosaccharide-forming enzyme means the unit(s) assayed asabove.

The α-isomaltosyl-transferring enzyme was assayed for enzymatic activityby dissolving panose in 100 mM acetate buffer (pH 6.0) to give aconcentration of 2% (w/v) for a substrate solution, adding a 0.5 ml ofan enzyme solution to 0.5 ml of the substrate solution, enzymaticallyreacting the mixture solution at 35° C. for 30 min, suspending thereaction mixture by boiling for 10 min, and quantifying glucose, amongthe cyclotetrasaccharide and glucose formed in the reaction mixture, bythe glucose oxidase method. One unit activity of theα-isomaltosyl-transferring enzyme is defined as the enzyme amount thatforms one micromole of glucose per minute under the above enzymaticreaction conditions. Throughout the specification, the enzymaticactivity of the α-isomaltosyl-transferring enzyme means the unit(s)assayed as above.

The cyclotetrasaccharide-forming activity is assayed by dissolving“PINE-DEX #100”, a partial starch hydrolysate commercialized byMatsutani Chemical Ind., Tokyo, Japan, in 50 mM acetate buffer (pH 6.0)to give a concentration of 2% (w/v) for a substrate solution, adding 0.5ml of an enzyme solution to 0.5 ml of the substrate solution,enzymatically reacting the mixture solution at 35° C. for 60 min,suspending the reaction mixture by boiling for 10 min, and then furtheradding to the resulting mixture one milliliter of 50 mM acetate buffer(pH 5.0) with 70 units/ml of “TRANSGLUCOSIDASE L AMANO™”, anα-glucosidase commercialized by Amano Pharmaceutical Co., Ltd., Aichi,Japan, and 27 units/ml of glucoamylase, commercialized by NagaseBiochemicals, Ltd., Kyoto, Japan, and incubated at 50° C. for 60 min,inactivating the remaining enzymes by heating at 100° C. for 10 min, andquantifying cyclotetrasaccharide on HPLC similarly as in Experiment 1.One unit of cyclotetrasaccharide-forming activity is defined as theenzyme amount that forms one micromole of cyclotetrasaccharide perminute under the above enzymatic reaction conditions. Throughout thespecification, the cyclotetrasaccharide-forming activity means theactivity (units) assayed as above.

EXPERIMENT 4 Preparation of Enzyme from Bacillus globisporus C9EXPERIMENT 4-1

About 18 L of the supernatant in Experiment 3 was salted out with 80%saturated ammonium sulfate and allowed to stand at 4° C. for 24 hours,and the formed sediments were collected by centrifugation at 10,000 rpmfor 30 min, dissolved in 10 mM phosphate buffer (pH 7.5), and dialyzedagainst a fresh preparation of the same buffer to obtain about 400 ml ofa crude enzyme solution with 8,110 units of theα-isomaltosylglucosaccharide-forming enzyme, 24,700 units ofα-isomaltosyl-transferring enzyme, and about 15,600 units ofcyclotetrasaccharide-forming activity. The crude enzyme solution wassubjected to ion-exchange chromatography using 1,000 ml of “SEPABEADSFP-DA13” gel, an ion-exchange resin commercialized by MitsubishiChemical Industries, Ltd., Tokyo, Japan. Theα-isomaltosylglucosaccharide-forming enzyme and cyclotetrasaccharidewere eluted as non-adsorbed fractions without adsorbing on theion-exchange resin. The resulting enzyme solution was dialyzed against10 mM phosphate buffer (pH 7.0) with 1 M ammonium sulfate, and thedialyzed solution was centrifuged to remove impurities, and subjected toaffinity chromatography using 500 ml of “SEPHACRYL HR S-200”, a gelcommercialized by Amersham Corp., Div. Amersham International, ArlingtonHeights, Ill., USA. Enzymatically active components adsorbed on the geland, when sequentially eluted with a linear gradient decreasing from 1 Mto 0 M of ammonium sulfate and a linear gradient increasing from 0 mM to100 mM of maltotetraose, the α-isomaltosylglucosaccharide-forming enzymeand the α-isomaltosyl-transferring enzyme were separately eluted, i.e.,the former was eluted with the linear gradient of maltotetraose at about30 mM and the latter was eluted with the linear gradient of ammoniumsulfate at about 0 M. Thus, fractions with α-isomaltosyl-transferringactivity and those with the α-isomaltosylglucosaccharide-formingactivity according to the present invention were separatory collected.No cyclotetrasaccharide-forming activity was found in any of the abovefractions and this revealed that a mixture solution of the abovefractions with α-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme had also cyclotetrasaccharide-formingactivity, and revealed that the activity of forming cyclotetrasaccharidefrom partial starch hydrolyzates was exerted by the coaction of theactivities of the above two types of enzymes.

Methods for separatory purifying theα-isomaltosylglucosaccharide-forming enzyme of the present invention andα-isomaltosyl-transferring enzyme are described in the below:

EXPERIMENT 4-2 Purification of α-isomaltosylglucosaccharide-formingEnzyme

A fraction of the α-isomaltosylglucosaccharide-forming enzyme of thepresent invention, obtained in Experiment 4-1, was dialyzed against 10mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate. Thedialyzed solution was centrifuged to remove insoluble impurities, andthe resulting supernatant was fed to hydrophobic chromatography using350 ml of “BUTYL-TOYOPEARL 650 M”, a gel commercialized by TosohCorporation, Tokyo, Japan. The enzyme was adsorbed on the gel and elutedat about 0.3 M ammonium sulfate when eluted with a linear gradientdecreasing from 1 M to 0 M of ammonium sulfate, followed by collectingfractions with the enzyme activity. The fractions were pooled and againdialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammoniumsulfate. The resulting dialyzed solution was centrifuged to removeimpurities and fed to affinity chromatography using “SEPHACRYL HR S-200”gel to purify the enzyme. The amount of enzyme activity, specificactivity, and yield of the α-isomaltosylglucosaccharide-forming enzymein each purification step are in Table 1. TABLE 1 Specific activityEnzyme* activity of enzyme* Yield Purification step (unit) (unit/mgprotein) (%) Culture supernatant 8,110 0.12 100 Dialyzed solution after7,450 0.56 91.9 salting out with ammonium sulfate Eluate fromion-exchange 5,850 1.03 72.1 column chromatography Eluate from affinity4,040 8.72 49.8 column chromatography Eluate from hydrophobic 3,070 10.637.8 column chromatography Eluate from affinity 1,870 13.6 23.1 columnchromatographyNote:The symbol “*” means the α-isomaltosylglucosaccharide-forming enzyme ofthe present invention.

The finally purified α-isomaltosylglucosaccharide-forming enzymespecimen was assayed for purity on gel electrophoresis using a 7.5%(w/v) polyacrylamide gel and detected on the gel as a single proteinband, i.e., a high purity enzyme specimen.

EXPERIMENT 4-3 Purification of α-isomaltosyl-transferring Enzyme

A fraction with α-isomaltosyl-transferring enzyme, which had beenseparated from a fraction with α-isomaltosylglucosaccharide-formingenzyme by affinity chromatography in Experiment 4-1, was dialyzedagainst 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate.The resulting dialyzed solution was centrifuged to remove impurities,and subjected to affinity chromatography using “SEPHACRYL HR S-200” gelto purify the enzyme. The amount of enzyme activity, specific activity,and yield of the α-isomaltosyl-transferring enzyme in each purificationstep are in Table 2. TABLE 2 Specific activity Enzyme* activity ofenzyme* Yield Purification step (unit) (unit/mg protein) (%) Culturesupernatant 26,900 0.41 100 Dialyzed solution after 24,700 1.85 91.8salting out with ammonium sulfate Eluate from ion-exchange 19,400 3.4172.1 column chromatography Eluate from affinity 13,400 18.6 49.8 columnchromatography Eluate from hydrophobic 10,000 21.3 37.2 columnchromatography Eluate from affinity 6,460 26.9 24.0 columnchromatographyNote:The symbol “*” means the α-isomaltosyl-transferring enzyme of thepresent invention.

EXPERIMENT 5 Property of α-isomaltosylglucosaccharide-forming Enzyme andα-isomaltosyl-transferring Enzyme EXPERIMENT 5-1 Property ofα-isomaltosylglucosaccharide-forming Enzyme

A purified specimen of α-isomaltosylglucosaccharide-forming enzyme,obtained by the method in Experiment 4-2, was subjected to SDS-PAGEusing a 7.5% (w/v) of polyacrylamide gel and then determined formolecular weight by comparing with the dynamics of standard molecularmarkers electrophoresed in parallel, commercialized by Bio-RadLaboratories Inc., Brussels, Belgium, revealing that the enzyme had amolecular weight of about 140,000∀20,000 daltons.

A fresh preparation of the above purified specimen was subjected toisoelectrophoresis using a gel containing 2% (w/v) ampholinecommercialized by Amersham Corp., Div. Amersham International, ArlingtonHeights, Ill., USA, and then measured for pHs of protein bands and gelto determine the isoelectric point of the enzyme, revealing that theenzyme had an isoelectric point of about 5.2∀0.5.

The influence of temperature and pH on the activity ofα-isomaltosylglucosaccharide-forming enzyme was examined in accordancewith the assay for the enzyme activity, where the influence oftemperature was conducted in the presence or absence of 1 mM Ca²⁺. Theseresults are in FIG. 5 (influence of temperature) and FIG. 6 (influenceof pH). The optimum temperature of the enzyme was about 40° C. (in theabsence of Ca²⁺) and about 45° C. (in the presence of 1 mM Ca²⁺) whenincubated at pH 6.0 for 60 min, and the optimum pH of the enzyme wasabout 6.0 to about 6.5 when incubated at 35° C. for 60 min. The thermalstability of the enzyme was determined by incubating the testing enzymesolutions in 20 mM acetate buffer (pH 6.0) at prescribed temperaturesfor 60 min in the presence or absence of 1 mM Ca²⁺, cooling with waterthe resulting enzyme solutions, and assaying the remaining enzymeactivity of each solution. The pH stability of the enzymes wasdetermined by keeping the testing enzyme solutions in 50 mM buffershaving prescribed pHs at 4° C. for 24 hours, adjusting the pH of eachsolution to 6.0, and assaying the remaining enzyme activity of eachsolution. These results are respectively in FIG. 7 (thermal stability)and FIG. 8 (pH stability). As a result, the enzyme had thermal stabilityof up to about 35° C. in the absence of Ca²⁺ and about 40° C. in thepresence of 1 mM Ca²⁺, and pH stability of about 4.5 to about 9.0.

The influence of metal ions on the activity ofα-isomaltosylglucosaccharide-forming enzyme was examined in the presenceof 1 mM of each metal-ion according to the assay for the enzymeactivity. The results are in Table 3. TABLE 3 Metal ion Relativeactivity (%) Metal ion Relative activity (%) None 100 Hg²⁺ 4 Zn²⁺ 92Ba²⁺ 65 Mg²⁺ 100 Sr²⁺ 80 Ca²⁺ 115 Pb²⁺ 103 Co²⁺ 100 Fe²⁺ 98 Cu²⁺ 15 Fe³⁺97 Ni²⁺ 98 Mn²⁺ 111 Al³⁺ 99 EDTA 20

As evident from the results in Table 3, the enzyme activity was greatlyinhibited by Hg²⁺, Cu²⁺, and EDTA, and was also inhibited by Ba²⁺ andSr²⁺. It was also found that the enzyme was activated by Ca²⁺ and Mn²⁺.

Amino acid analysis of the N-terminal amino acid sequence of the enzymeby “PROTEIN SEQUENCER MODEL 473A”, an apparatus of Applied Biosystems,Inc., Foster City, USA, revealed that the enzyme had a partial aminoacid sequence of SEQ ID NO:1, i.e.,tyrosine-valine-serine-serine-leucine-glycine-asparagine-leucine-isoleucinein the N-terminal region.

EXPERIMENT 5-2 Property of α-isomaltosyl-transferring Enzyme

A purified specimen of α-isomaltosyl-transferring enzyme, obtained bythe method in Experiment 4-3, was subjected to SDS-PAGE using a 7.5%(w/v) of polyacrylamide gel and then determined for molecular weight bycomparing with the dynamics of standard molecular markerselectrophoresed in parallel, commercialized by Bio-Rad LaboratoriesInc., Brussels, Belgium, revealing that the enzyme had a molecularweight of about 112,000∀20,000 daltons.

A fresh preparation of the above purified specimen was subjected toisoelectrophoresis using a gel containing 2% (w/v) ampholinecommercialized by Amersham Corp., Div. Amersham International, ArlingtonHeights, Ill., USA, and then measured for pHs of protein bands and gelto determine the isoelectric point of the enzyme, revealing that theenzyme had an isoelectric point of about 5.5∀0.5.

The influence of temperature and pH on the activity ofα-isomaltosyl-transferring enzyme was examined in accordance with theassay for the enzyme activity. These results are in FIG. 9 (influence oftemperature) and FIG. 10 (influence of pH). The optimum temperature ofthe enzyme was about 45° C. when incubated at pH 6.0 for 30 min, and theoptimum pH of the enzyme was about 6.0 when incubated at 35° C. for 30min. The thermal stability of the enzyme was determined by incubatingthe testing enzyme solutions in 20 mM acetate buffer (pH 6.0) atprescribed temperatures for 60 min, cooling with water the resultingenzyme solutions, and assaying the remaining enzyme activity of eachsolution. The pH stability of the enzyme was determined by keeping thetesting enzyme solutions in 50 mM buffers having prescribed pHs at 4° C.for 24 hours, adjusting the pH of each solution to 6.0, and assaying theremaining enzyme activity of each solution. These results arerespectively in FIG. 11 (thermal stability) and FIG. 12 (pH stability).As a result, the enzyme had thermal stability of up to about 40° C. andpH stability of about 4.0 to about 9.0.

The influence of metal ions on the activity ofα-isomaltosylglucosaccharide forming enzyme was examined in the presenceof 1 mM of each metal-ion according to the assay for the enzymeactivity. The results are in Table 4. TABLE 4 Metal ion Relativeactivity (%) Metal ion Relative activity (%) None 100 Hg²⁺ 1 Zn²⁺ 88Ba²⁺ 102 Mg²⁺ 98 Sr²⁺ 101 Ca²⁺ 101 Pb²⁺ 89 Co²⁺ 103 Fe²⁺ 96 Cu²⁺ 57 Fe³⁺105 Ni²⁺ 102 Mn²⁺ 106 Al³⁺ 103 EDTA 104

As evident from the results in Table 4, the enzyme activity was greatlyinhibited by Hg²⁺ and was also inhibited by Cu²⁺. It was also found thatthe enzyme was not activated by Ca²⁺ and not inhibited by EDTA.

Amino acid analysis of the N-terminal amino acid sequence of the enzymeby “PROTEIN SEQUENCER MODEL 473A”, an apparatus of Applied Biosystems,Inc., Foster City, USA, revealed that the enzyme had a partial aminoacid sequence of SEQ ID NO:2, i.e, isoleucine-asparticacid-glycine-valine-tyrosine-histidine-alanine-proline-asparagine-glycinein the N-terminal region.

EXPERIMENT 6 Production of α-isomaltosylglucosaccharide-forming Enzymefrom Bacillus globisporus C11 Strain C11

A liquid nutrient culture medium, consisting of 4.0% (w/v) of “PINE-DEX#100”, a partial starch hydrolysate, 1.8% (w/v) of “ASAHIMEAST”, a yeastextract, 0.1% (w/v) of dipotassium phosphate, 0.06% (w/v) of sodiumphosphate dodecahydrate, 0.05% (w/v) magnesium sulfate heptahydrate, andwater was placed in 500-ml Erlenmeyer flasks in a volume of 100 ml each,autoclaved at 121° C. for 20 minutes to effect sterilization, cooled,inoculated with a stock culture of Bacillus globisporus C11, FERMBP-7144, and incubated at 27° C. for 48 hours under rotary shakingconditions of 230 rpm. The resulting cultures were pooled and used as aseed culture.

About 20 L of a fresh preparation of the same nutrient culture medium asused in the above culture were placed in a 30-L fermentor, sterilized byheating, cooled to 27° C., inoculated with 1% (v/v) of the seed culture,and incubated for about 48 hours while stirring under aeration agitationconditions at 27° C. and pH 6.0-8.0. The resultant culture, having about0.55 unit/ml of α-isomaltosylglucosaccharide-forming enzyme activity,about 1.8 units/ml of α-isomaltosyl-transferring enzyme activity, andabout 1.1 units/ml of cyclotetrasaccharide-forming enzyme activity, wascentrifuged at 10,000 rpm for 30 min to obtain about 18 L of asupernatant. Measurement of the supernatant revealed that it had about0.51 unit/ml of α-isomaltosylglucosaccharide-forming enzyme activity,i.e., a total enzyme activity of about 9,180 units; about 1.7 units/mlof α-isomaltosyl-transferring enzyme activity, i.e., a total enzymeactivity of about 30,400 units; and about 1.1 units/ml ofcyclotetrasaccharide-forming enzyme activity, i.e., a total enzymeactivity of about 19,400 units.

EXPERIMENT 7 Preparation of Enzyme from Bacillus globisporus C11

An 18 L of the supernatant obtained in Experiment 6 was salted out withan 80% saturated ammonium sulfate solution and allowed to stand at 4° C.for 24 hours. Then the salted out sediments were collected bycentrifugation at 10,000 for 30 min, dissolved in 10 mM phosphate buffer(pH 7.5), dialyzed against a fresh preparation of the same buffer toobtain about 416 ml of a crude enzyme solution. The crude enzymesolution was revealed to have 8,440 units of theα-isomaltosylglucosaccharide-forming enzyme, about 28,000 units ofα-isomaltosyl-transferring enzyme, and about 17,700 units ofcyclotetrasaccharide-forming enzyme. When subjected to ion-exchangechromatography using “SEPABEADS FP-DA13” gel, disclosed in Experiment4-1, the above three types of enzymes were eluted as non-adsorbedfractions without adsorbing on the gel. The non-adsorbed fractions withthose enzymes were pooled and dialyzed against 10 mM phosphate buffer(pH 7.0) containing 1 M ammonium sulfate, and the dialyzed solution wascentrifuged to remove impurities. The resulting supernatant was fed toaffinity chromatography using 500 ml of “SEPHACRYL HR S-200” gel topurify the enzyme. Active enzymes was adsorbed on the gel andsequentially was eluted with a linear gradient decreasing from 1 M to 0M of ammonium sulfate and a linear gradient increasing from 0 mM to 100mM of maltotetraose, followed by separate elution ofα-isomaltosyl-transferring enzyme and theα-isomaltosylglucosaccharide-forming enzyme, where the former enzyme waseluted with the linear gradient of ammonium sulfate at a concentrationof about 0.3 M and the latter enzyme was eluted with a linear gradientof maltotetraose at a concentration of about 30 mM. Therefore, fractionswith the α-isomaltosylglucosaccharide-forming enzyme of the presentinvention and those with α-isomaltosyl-transferring enzyme wereseparately collected and recovered. Similarly as in the case of Bacillusglobisporus C9 in Experiment 4, it was found that nocyclotetrasaccharide-forming activity was found in any fraction in thiscolumn chromatography, and that an enzyme mixture solution of bothfractions of α-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme showed cyclotetrasaccharide-formingactivity, revealing that the activity of forming cyclotetrasaccharidefrom partial starch hydrolyzates was exerted in collaboration with theenzyme activities of these enzymes.

The methods for separately purifying theα-isomaltosylglucosaccharide-forming enzyme of the present invention andα-isomaltosyl-transferring enzyme are disclosed hereinafter:

EXPERIMENT 7-2 Purification of α-isomaltosylglucosaccharide-formingEnzyme

A fraction of the α-isomaltosylglucosaccharide-forming enzyme of thepresent invention was dialyzed against 10 mM phosphate buffer (pH 7.0)containing 1 M ammonium sulfate. The dialyzed solution was centrifugedto remove insoluble impurities, and the resulting supernatant was fed tohydrophobic chromatography using 350 ml of “BUTYL-TOYOPEARL 650 M”, agel commercialized by Tosoh Corporation, Tokyo, Japan. The enzymeadsorbed on the gel was eluted at about 0.3 M ammonium sulfate wheneluted with a linear gradient decreasing from 1 M to 0 M of ammoniumsulfate, followed by collecting fractions with the enzyme activity. Thefractions were pooled and dialyzed against 10 mM phosphate buffer (pH7.0) containing 1 M ammonium sulfate. The resulting dialyzed solutionwas centrifuged to remove impurities and fed to affinity chromatographyusing “SEPHACRYL HR S-200” gel to purify the enzyme. The amount ofenzyme activity, specific activity, and yield of theα-isomaltosylglucosaccharide-forming enzyme in each purification stepare in Table 5. TABLE 5 Specific activity Enzyme* activity of enzyme*Yield Purification step (unit) (unit/mg protein) (%) Culture supernatant9,180 0.14 100 Dialyzed solution after 8,440 0.60 91.9 salting out withammonium sulfate Eluate from ion-exchange 6,620 1.08 72.1 columnchromatography Eluate from affinity 4,130 8.83 45.0 columnchromatography Eluate from hydrophobic 3,310 11.0 36.1 columnchromatography Eluate from affinity 2,000 13.4 21.8 columnchromatographyNote:The symbol “*” means the α-isomaltosylglucosaccharide-forming enzyme ofthe present invention.

The finally purified α-isomaltosylglucosaccharide-forming enzymespecimen was assayed for purity on gel electrophoresis using a 7.5%(w/v) polyacrylamide gel and detected on the gel as a single proteinband, meaning a high purity enzyme specimen.

EXPERIMENT 7-3 Purification of α-isomaltosyl-transferring Enzyme

A fraction of α-isomaltosyl-transferring enzyme, which had beenseparated from a fraction with α-isomaltosylglucosaccharide-formingenzyme by the affinity chromatography in Experiment 7-1, was dialyzedagainst 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate.The dialyzed solution was centrifuged to remove insoluble impurities,and the resulting supernatant was fed to hydrophobic chromatographyusing 350 ml of “BUTYL-TOYOPEARL 650 M”, a gel commercialized by TosohCorporation, Tokyo, Japan. The enzyme adsorbed on the gel and then itwas eluted at about 0.3 M ammonium sulfate when eluted with a lineargradient decreasing from 1 M to 0 M of ammonium sulfate, followed bycollecting fractions with the enzyme activity. The fractions were pooledand dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 Mammonium sulfate. The resulting dialyzed solution was centrifuged toremove impurities and fed to affinity chromatography using “SEPHACRYL HRS-200” gel to purify the enzyme. The amount of enzyme activity, specificactivity, and yield of the α-isomaltosyl-transferring enzyme in eachpurification step are in Table 6. TABLE 6 Specific activity Enzyme*activity of enzyme* Yield Purification step (unit) (unit/mg protein) (%)Culture supernatant 30,400 0.45 100 Dialyzed solution after 28,000 1.9892.1 salting out with ammonium sulfate Eluate from ion-exchange 21,8003.56 71.7 column chromatography Eluate from affinity 13,700 21.9 45.1column chromatography Eluate from hydrophobic 10,300 23.4 33.9 columnchromatography Eluate from affinity 5,510 29.6 18.1 columnchromatographyNote:The symbol “*” means α-isomaltosyl-transferring enzyme.

EXPERIMENT 8 Property of α-isomaltosylglucosaccharide-forming Enzyme andα-isomaltosyl-transferring Enzyme EXPERIMENT 8-1 Property ofα-isomaltosylglucosaccharide-forming Enzyme

A purified specimen of α-isomaltosylglucosaccharide-forming enzyme,obtained by the method in Experiment 7-2, was subjected to SDS-PAGEusing a 7.5% (w/v) of polyacrylamide gel and then determined formolecular weight by comparing with the dynamics of standard molecularmarkers electrophoresed in parallel, commercialized by Bio-RadLaboratories Inc., Brussels, Belgium, revealing that the enzyme had amolecular weight of about 137,000∀20,000 daltons.

A fresh preparation of the above purified specimen was subjected toisoelectrophoresis using a gel containing 2% (w/v) ampholinecommercialized by Amersham Corp., Div. Amersham International, ArlingtonHeights, Ill., USA, and then measured for pHs of protein bands and gelto determine the isoelectric point of the enzyme, revealing that theenzyme had an isoelectric point of about 5.2∀0.5.

The influence of temperature and pH on the activity ofα-isomaltosylglucosaccharide-forming enzyme was examined in accordancewith the assay for the enzyme activity, where the influence oftemperature was conducted in the presence or absence of 1 mM Ca²⁺. Theseresults are in FIG. 13 (influence of temperature) and FIG. 14 (influenceof pH). The optimum temperature of the enzyme was about 45° C. in theabsence of Ca²⁺ and about 50° C. in the presence of 1 mM Ca²⁺ whenincubated at pH 6.0 for 60 min. The optimum pH of the enzyme was about6.0 when incubated at 35° C. for 60 min. The thermal stability of theenzyme was determined by incubating the testing enzyme solutions in 20mM acetate buffer (pH 6.0) in the presence or absence of 1 mM Ca²⁺ atprescribed temperatures for 60 min, cooling with water the resultingenzyme solutions, and assaying the remaining enzyme activity of eachsolution. The pH stability of the enzyme was determined by keeping thetesting enzyme solutions in 50 mM buffers having prescribed pHs at 4° C.for 24 hours, adjusting the pH of each solution to 6.0, and assaying theremaining enzyme activity of each solution. These results arerespectively in FIG. 15 (thermal stability) and FIG. 16 (pH stability).As a result, the enzyme had thermal stability of up to about 40° C. inthe absence of Ca²⁺ and up to about 45° C. in the presence of 1 mM Ca²⁺.The pH stability of enzyme was about 5.0 to about 10.0.

The influence of metal ions on the activity ofα-isomaltosylglucosaccharide forming enzyme was examined in the presenceof 1 mM of each metal-ion according to the assay for the enzymeactivity. The results are in Table 7. TABLE 7 Metal ion Relativeactivity (%) Metal ion Relative activity (%) None 100 Hg²⁺ 4 Zn²⁺ 91Ba²⁺ 65 Mg²⁺ 98 Sr²⁺ 83 Ca²⁺ 109 Pb²⁺ 101 Co²⁺ 96 Fe²⁺ 100 Cu²⁺ 23 Fe³⁺102 Ni²⁺ 93 Mn²⁺ 142 Al³⁺ 100 EDTA 24

As evident from the results in Table 7, the enzyme activity was greatlyinhibited by Hg²⁺, Cu²⁺, and EDTA and was also inhibited by Ba²⁺ andSr²⁺. It was also found that the enzyme was activated by Ca²⁺ and Mn²⁺.

Amino acid analysis of the N-terminal amino acid sequence of the enzymeby “PROTEIN SEQUENCER MODEL 473A”, an apparatus of Applied Biosystems,Inc., Foster City, USA, revealed that the enzyme had a partial aminoacid sequence of SEQ ID NO:1, i.e,tyrosine-valine-serine-serine-leucine-glycine-asparagine-leucine-isoleucinein the N-terminal region.

The comparison of the partial amino acid sequence in the N-terminalregion with that derived from the α-isomaltosylglucosaccharide-formingenzyme from Bacillus globisporus C9 in Experiment 5-1 revealed that theywere the same and the N-terminal amino acid sequence, commonly found inα-isomaltosylglucosaccharide-forming enzymes, was an amino acid sequenceoftyrosine-valine-serine-serine-leucine-glycine-asparagine-leucine-isoleucineof SEQ ID NO:1 in the N-terminal region.

EXPERIMENT 8-2 Property of α-isomaltosyl-transferring Enzyme

A purified specimen of α-isomaltosyl-transferring enzyme, obtained bythe method in Experiment 7-3, was subjected to SDS-PAGE using a 7.5%(w/v) of polyacrylamide gel and then determined for molecular weight bycomparing with the dynamics of standard molecular markerselectrophoresed in parallel, commercialized by Bio-Rad LaboratoriesInc., Brussels, Belgium, revealing that the enzyme had a molecularweight of about 102,000∀20,000 daltons.

A fresh preparation of the above purified specimen was subjected toisoelectrophoresis using a gel containing 2% (w/v) ampholinecommercialized by Amersham Corp., Div. Amersham International, ArlingtonHeights, Ill., USA, and then measured for pHs of protein bands and gelto determine the isoelectric point of the enzyme, revealing that theenzyme had an isoelectric point of about 5.6∀0.5.

The influence of temperature and pH on the activity ofα-isomaltosyl-transferring enzyme was examined in accordance with theassay for the enzyme activity. These results are in FIG. 17 (influenceof temperature) and FIG. 18 (influence of pH). The optimum temperatureof the enzyme was about 50° C. when incubated at pH 6.0 for 30 min. Theoptimum pH of the enzyme was about 5.5 to about 6.0 when incubated at35° C. for 30 min. The thermal stability of the enzyme was determined byincubating the testing enzyme solutions in 20 mM acetate buffer (pH 6.0)at prescribed temperatures for 60 min, cooling with water the resultingenzyme solutions, and assaying the remaining enzyme activity of eachsolution. The pH stability of the enzyme was determined by keeping thetesting enzyme solutions in 50 mM buffers having prescribed pHs at 4° C.for 24 hours, adjusting the pH of each solution to 6.0, and assaying theremaining enzyme activity of each solution. These results arerespectively in FIG. 19 (thermal stability) and FIG. 20 (pH stability).As a result, the enzyme had thermal stability of up to about 40° C. andpH stability of about 4.5 to about 9.0.

The influence of metal ions on the activity ofα-isomaltosyl-transferring enzyme was examined in the presence of 1 mMof each metal-ion according to the assay for the enzyme activity. Theresults are in Table 8. TABLE 8 Relative Relative Metal ion activity (%)Metal ion activity (%) None 100 Hg²⁺ 2 Zn²⁺ 83 Ba²⁺ 90 Mg²⁺ 91 Sr²⁺ 93Ca²⁺ 91 Pb²⁺ 74 Co²⁺ 89 Fe²⁺ 104 Cu²⁺ 56 Fe³⁺ 88 Ni²⁺ 89 Mn²⁺ 93 Al³⁺ 89EDTA 98

As evident from the results in Table 8, the enzyme activity was greatlyinhibited by Hg²⁺ and was also inhibited by Cu²⁺. It was also found thatthe enzyme was not activated by Ca²⁺ and not inhibited by EDTA.

Amino acid analysis of the N-terminal amino acid sequence of the enzymeby “PROTEIN SEQUENCER MODEL 473A”, an apparatus of Applied Biosystems,Inc., Foster City, USA, revealed that the enzyme had a partial aminoacid sequence of SEQ ID NO:3, i.e., isoleucine-asparticacid-glycine-valine-tyrosine-histidine-alanine-proline-tyrosine-glycinein the N-terminal region.

The comparison of the partial amino acid sequence in the N-terminalregion with that derived from the α-isomaltosyl-transferring enzyme fromBacillus globisporus C9 in Experiment 5-2 revealed that they had acommon amino acid sequence of isoleucine-asparticacid-glycine-valine-tyrosine-histidine-alanine-proline, as shown in SEQID NO:4 at their N-terminal regions.

EXPERIMENT 9 Amino Acid Sequence of α-isomaltosylglucosaccharide-formingEnzyme and α-isomaltosyl-transferring Enzyme EXPERIMENT 9-1 InternalAmino Acid Sequence of α-isomaltosylglucosaccharide-forming Enzyme

A part of a purified specimen of α-isomaltosylglucosaccharide-formingenzyme, obtained by the method in Experiment 7-2, was dialyzed against10 mM Tris-HCl buffer (pH 9.0), and the dialyzed solution was dilutedwith a fresh preparation of the same buffer to give a concentration ofabout one milligram per milliliter. One milliliter of the dilute as atest sample was admixed with 10 Φg of trypsin commercialized by WakoPure Chemical Industries, Ltd., Tokyo, Japan, and incubated at 30° C.for 22 hours to hydrolyze into peptides. To isolate the hydrolyzedpeptides, the resulting hydrolyzates were subjected to reverse-phaseHPLC using “Φ-Bondapak C18 column” with a diameter of 2.1 mm and alength of 150 mm, a product of Waters Chromatography Div., MILLIPORECorp., Milford, USA, at a flow rate of 0.9 ml/min and at ambienttemperature, and using a liner gradient of acetonitrile increasing from8% (v/v) to 40% (v/v) in 0.1% (v/v) trifluoroacetate over 120 min. Thepeptides eluted from the column were detected by monitoring theabsorbency at a wavelength of 210 nm. Three peptide specimens named P64with a retention time of about 64 min, P88 with a retention time ofabout 88 min, and P99 with a retention time of about 99 min, which hadbeen well separated from other peptides, were separately collected anddried in vacuo and then dissolved in 200 Φl of a solution of 0.1% (v/v)trifluoroacetate and 50% (v/v) acetonitrile. Each peptide specimen wassubjected to a protein sequencer for analyzing amino acid sequence up toeight amino acid residues to obtain amino acid sequences of SEQ ID NOs:5to 7. The analyzed internal partial amino acid sequences are in Table 9.TABLE 9 Peptide name Internal partial amino acid sequence P64 asparticacid-alanine-serine-alanine- SEQ ID NO: 5asparagine-valine-threonine-threonine P88tryptophan-serine-leucine-glycine- SEQ ID NO: 6phenylalanine-methionine-asparagine- phenylalanine P99asparagine-tyrosine-threonine-aspartic SEQ ID NO: 7acid-alanine-tryptophan-methionine- phenylalanine

EXPERIMENT 9-2 Internal Amino Acid Sequence ofα-isomaltosyl-transferring Enzyme

A part of a purified specimen of α-isomaltosyl-transferring enzyme,obtained by the method in Experiment 7-3, was dialyzed against 10 mMTris-HCl buffer (pH 9.0), and the dialyzed solution was diluted with afresh preparation of the same buffer to give a concentration of aboutone milligram per milliliter. One milliliter of the dilute as a testsample was admixed with 10 Φg of “Lysyl Endopeptidase” commercialized byWako Pure Chemical Industries, Ltd., Tokyo, Japan, and allowed to reactat 30° C. for 22 hours to form peptides. The resultant mixtures weresubjected to reverse-phase HPLC to separate the peptides using“Φ-Bondapak C18 column” having a diameter of 2.1 mm and a length of 150mm, a product of Waters Chromatography Div., MILLIPORE Corp., Milford,USA, at a flow rate of 0.9 ml/min and at ambient temperature, and usinga liner gradient of acetonitrile increasing from 8% (v/v) to 40% (v/v)in 0.1% (v/v) trifluoroacetate over 120 min. The peptides eluted fromthe column were detected by monitoring the absorbency at a wavelength of210 nm. Three peptide specimens named P22 with a retention time of about22 min, P63 with a retention time of about 63 min, and P71 with aretention time of about 71 min, which had been well separated from otherpeptides, were separately collected and dried in vacuo and thendissolved in 200 Φl of a solution of 0.1% (v/v) trifluoroacetate and 50%(v/v) acetonitrile. Each peptide specimen was subjected to a proteinsequencer for analyzing amino acid sequence up to eight amino acidresidues to obtain amino acid sequences of SEQ ID NOs:8 to 10. Theanalyzed internal partial amino acid sequences are in Table 10. TABLE 10Peptide name Internal partial amino acid sequence P22glycine-asparagine-glutamic acid-methionine- SEQ ID NO: 8arginine-asparagine-glutamine-tyrosine P63isoleucine-threonine-threonine- SEQ ID NO: 9tryptophan-proline-isoleucine- glutamic acid-serine P71tryptophan-alanine-phenylalanine-glycine- SEQ IDleucine-tryptophan-methionine-serine NO: 10

EXPERIMENT 10 Production of α-isomaltosylglucosaccharide-forming Enzymefrom Bacillus globisporus N75 Strain N75

A liquid nutrient culture medium, consisting of 4.0% (w/v) of “PINE-DEX#4”, a partial starch hydrolysate, 1.8% (w/v) of “ASAHIMEAST”, a yeastextract, 0.1% (w/v) of dipotassium phosphate, 0.06% (w/v) of sodiumphosphate dodecahydrate, 0.05% (w/v) magnesium sulfate heptahydrate, andwater was placed in 500-ml Erlenmeyer flasks in a volume of 100 ml each,autoclaved at 121° C. for 20 minutes to effect sterilization, cooled,inoculated with a stock culture of Bacillus globisporus N75, FERMBP-7591, and incubated at 27° C. for 48 hours under rotary shakingconditions of 230 rpm for use as a seed culture.

About 20 L of a fresh preparation of the same nutrient culture medium asused in the above culture were placed in a 30-L fermentor, sterilized byheating, cooled to 27° C., inoculated with 1% (v/v) of the seed culture,and incubated for about 48 hours while stirring under aeration agitationconditions at 27° C. and pH 6.0-8.0. The resultant culture, having about0.34 unit/ml of α-isomaltosylglucosaccharide-forming enzyme activity,about 1.1 units/ml of α-isomaltosyl-transferring enzyme activity, andabout 0.69 unit/ml of cyclotetrasaccharide-forming enzyme activity, wascentrifuged at 10,000 rpm for 30 min to obtain about 18 L of asupernatant. Measurement of the supernatant revealed that it had about0.33 unit/ml of α-isomaltosylglucosaccharide-forming enzyme activity,i.e., a total enzyme activity of about 5,940 units; about 1.1 units/mlof α-isomaltosyl-transferring enzyme activity, i.e., a total enzymeactivity of about 19,800 units; and about 0.67 unit/ml ofcyclotetrasaccharide-forming enzyme activity, i.e., a total enzymeactivity of about 12,100 units.

EXPERIMENT 11 Preparation of Enzyme from Bacillus globisporus N75

An 18 L of the supernatant obtained in Experiment 10 was salted out witha 60% saturated ammonium sulfate solution and allowed to stand at 4° C.for 24 hours. Then, the salted out sediments were collected bycentrifugation at 10,000 for 30 min, dissolved in 10 mM Tris-HCl buffer(pH 8.3), dialyzed against a fresh preparation of the same buffer toobtain about 450 ml of a crude enzyme solution. The crude enzymesolution was revealed to have 4,710 units of theα-isomaltosylglucosaccharide-forming enzyme, about 15,700 units ofα-isomaltosyl-transferring enzyme, and about 9,590 units ofcyclotetrasaccharide-forming enzyme, followed by subjecting it toion-exchange chromatography using “SEPABEADS FP-DA13” gel, disclosed inExperiment 4-1. The enzyme was adsorbed on the gel, whileα-isomaltosyl-transferring enzyme was eluted as a non-adsorbed fractionwithout adsorption on the gel. When eluted with a linear gradientincreasing from 0 M to 1 M NaCl, theα-isomaltosylglucosaccharide-forming enzyme of the present invention waseluted at a concentration of about 0.25 M NaCl. Under these conditions,fractions with the α-isomaltosylglucosaccharide-forming enzyme activityof the present invention and those with α-isomaltosyl-transferringenzyme were separately fractionated and collected. Similarly as in thecase of Bacillus globisporus C9 in Experiment 4 and Bacillus globisporusC11 in Experiment 7, it was revealed that nocyclotetrasaccharide-forming activity was found in any fraction in thiscolumn chromatography, and an enzyme solution, obtained by mixing bothfractions of α-isomaltosylglucosaccharide-forming enzyme and ofα-isomaltosyl-transferring enzyme, showed cyclotetrasaccharide-formingactivity, and these facts revealed that the activity of formingcyclotetrasaccharide from partial starch hydrolyzates is exerted by thecoaction of the α-isomaltosylglucosaccharide-forming enzyme of thepresent invention and α-isomaltosyl-transferring enzyme.

The following experiments describe a method of separately purifying theα-isomaltosylglucosaccharide-forming enzyme of the present invention andα-isomaltosyl-transferring enzyme:

EXPERIMENT 11-2 Purification of α-isomaltosylglucosaccharide-formingEnzyme

The above fractions with the α-isomaltosylglucosaccharide-forming enzymeof the present invention were pooled and then dialyzed against 10 mMphosphate buffer (pH 7.0) containing 1 M ammonium sulfate, and thedialyzed solution was centrifuged to remove impurities and fed toaffinity chromatography using 500 ml of “SEPHACRYL HR S-200” gel. Theenzyme was adsorbed on the gel and then eluted therefrom sequentiallywith a linear gradient decreasing from 1 M to 0 M ammonium sulfate andwith a linear gradient increasing from 0 mM to 100 mM maltotetraose. Asa result, the α-isomaltosylglucosaccharide-forming enzyme adsorbed onthe gel was eluted therefrom at a concentration of about 30 mMmaltotetraose, followed by collecting fractions with the enzymeactivity. The fractions were pooled and dialyzed against 10 mM phosphatebuffer (pH 7.0) containing 1 M ammonium sulfate, and the dialyzedsolution was centrifuged to remove impurities. The resulting supernatantwas fed to hydrophobic chromatography using 350 ml of “BUTYL-TOYOPEARL650M”, a gel commercialized by Tosoh Corporation, Tokyo, Japan. Theenzyme was adsorbed on the gel and then eluted with a linear gradientdecreasing from 1 M to 0 M ammonium sulfate, resulting in an elution ofthe enzyme from the gel at a concentration of about 0.3 M ammoniumsulfate and collecting fractions with the enzyme activity. The fractionswere pooled and dialyzed against 10 mM phosphate buffer (pH 7.0)containing 1 M ammonium sulfate, and the dialyzed solution wascentrifuged to remove impurities and purified on affinity chromatographyusing 350 ml of “SEPHACRYL HR S-200” gel. The amount of enzyme activity,specific activity, and yield of the α-isomaltosylglucosaccharide-formingenzyme in each purification step are in Table 11. TABLE 11 Specificactivity Enzyme* activity of enzyme* Yield Purification step (unit)(unit/mg protein) (%) Culture supernatant 5,940 0.10 100 Dialyzedsolution after 4,710 0.19 79.3 salting out with ammonium sulfate Eluatefrom ion-exchange 3,200 2.12 53.9 column chromatography Eluate fromaffinity 2,210 7.55 37.2 column chromatography Eluate from hydrophobic1,720 10.1 29.0 column chromatography Eluate from affinity 1,320 12.522.2 column chromatographyNote:The symbol “*” means α-isomaltosylglucosaccharide-forming enzyme.

The final purified α-isomaltosylglucosaccharide-forming enzyme specimenwas assayed for purity on gel electrophoresis using a 7.5% (w/v)polyacrylamide gel and detected on the gel as a single protein band,meaning a high purity enzyme specimen.

EXPERIMENT 11-3 Purification of α-isomaltosyl-transferring Enzyme

Fractions of α-isomaltosyl-transferring enzyme, which had been separatedfrom fractions of α-isomaltosylglucosaccharide-forming enzyme byion-exchange chromatography in Experiment 11-1, were pooled and dialyzedagainst 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate,and the dialyzed solution was centrifuged to remove impurities. Theresulting supernatant was fed affinity column chromatography using 500ml of “SEPHACRYL HR S-200”, a gel commercialized by Amersham Corp., Div.Amersham International, Arlington Heights, Ill., USA. The enzyme wasadsorbed on the gel and then eluted with a linear gradient decreasingfrom 1 M to 0 M of ammonium sulfate, resulting in an elution of theenzyme from the gel at a concentration of about 0.3 M ammonium sulfateand collecting fractions with the enzyme activity. The fractions werepooled and dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1M ammonium sulfate, and the dialyzed solution was centrifuged to removeimpurities and purified on hydrophobic chromatography using 380 ml of“BUTYL-TOYOPEARL 650M” gel. The enzyme was adsorbed on the gel and theneluted therefrom with a linear gradient decreasing from 1 M to 0 Mammonium sulfate, resulting in an elution of the enzyme at aconcentration of about 0.3 M ammonium sulfate. The fractions with theenzyme activity were pooled and dialyzed against 10 mM Tris-HCl buffer(pH 8.0), and the dialyzed solution was centrifuged to removeimpurities. The resulting supernatant was fed to ion-exchange columnchromatography using 380 ml of “SUPER Q-TOYOPEARL 650C” gelcommercialized by Tosoh Corporation, Tokyo, Japan. The enzyme was notadsorbed on the gel and then eluted as non-adsorbed fractions which werethen collected and pooled to obtain a final purified enzyme preparation.The amount of enzyme activity, specific activity, and yield of theα-isomaltosylglucosaccharide-forming enzyme in each purification stepare in Table 12. TABLE 12 Specific activity Enzyme* activity of enzyme*Yield Purification step (unit) (unit/mg protein) (%) Culture supernatant19,000 0.33 100 Dialyzed solution after 15,700 0.64 82.6 salting outwith ammonium sulfate Eluate from ion-exchange 12,400 3.56 65.3 columnchromatography Eluate from affinity 8,320 11.7 43.8 columnchromatography Eluate from hydrophobic 4,830 15.2 25.4 columnchromatography Eluate from ion-exchange 3,850 22.6 20.3 columnchromatographyNote:The symbol “*” means α-isomaltosyl-transferring enzyme.

The final purified α-isomaltosyl-transferring enzyme specimen wasassayed for purity on gel electrophoresis using a 7.5% (w/v)polyacrylamide gel and detected on the gel as a single protein band,meaning a high purity enzyme specimen.

EXPERIMENT 12 Property of α-isomaltosylglucosaccharide-forming Enzymeand α-isomaltosyl-transferring Enzyme EXPERIMENT 12-1 Property ofα-isomaltosylglucosaccharide-forming Enzyme

A purified specimen of α-isomaltosylglucosaccharide-forming enzyme,obtained by the method in Experiment 11-2, was subjected to SDS-PAGEusing a 7.5% (w/v) of polyacrylamide gel and then determined formolecular weight by comparing with the dynamics of standard molecularmarkers electrophoresed in parallel, commercialized by Bio-RadLaboratories Inc., Brussels, Belgium, revealing that the enzyme had amolecular weight of about 136,000∀20,000 daltons.

A fresh preparation of the above purified specimen was subjected toisoelectrophoresis using a gel containing 2% (w/v) ampholinecommercialized by Amersham Corp., Div. Amersham International, ArlingtonHeights, Ill., USA, and then measured for pHs of protein bands and gelto determine the isoelectric point of the enzyme, revealing that theenzyme had an isoelectric point of about 7.3∀0.5.

The influence of temperature and pH on the activity ofα-isomaltosylglucosaccharide-forming enzyme was examined in accordancewith the assay for the enzyme activity, where the influence oftemperature was conducted in the presence or absence of 1 mM Ca²⁺. Theseresults are in FIG. 21 (influence of temperature) and FIG. 22 (influenceof pH). The optimum temperature of the enzyme was about 50° C. and about55° C. when incubated at pH 6.0 for 60 min in the absence of and in thepresence of 1 mM Ca²⁺, respectively. The optimum pH of the enzyme wasabout 6.0 when incubated at 35° C. for 60 min. The thermal stability ofthe enzyme was determined by incubating the testing enzyme solutions in20 mM acetate buffer (pH 6.0) at prescribed temperatures for 60 min inthe absence of and in the presence of 1 mM Ca²⁺, cooling with water theresulting enzyme solutions, and assaying the remaining enzyme activityof each solution. The pH stability of the enzyme was determined bykeeping the testing enzyme solutions in 50 mM buffers having prescribedpHs at 4° C. for 24 hours, adjusting the pH of each solution to 6.0, andassaying the remaining enzyme activity of each solution. These resultsare respectively in FIG. 23 (thermal stability) and FIG. 24 (pHstability). As a result, the enzyme had thermal stability of up to about45° C. and about 50° C. in the absence of and in the presence of 1 mMCa²⁺, respectively, and had pH stability of about 5.0 to about 9.0.

The influence of metal ions on the activity ofα-isomaltosylglucosaccharide-forming enzyme was examined in the presenceof 1 mM of each metal-ion according to the assay for the enzymeactivity. The results are in Table 13. TABLE 13 Relative Relativeactivity activity Metal ion (%) Metal ion (%) None 100 Hg²⁺ 1 Zn²⁺ 82Ba²⁺ 84 Mg²⁺ 96 Sr²⁺ 85 Ca²⁺ 108 Pb²⁺ 86 Co²⁺ 93 Fe²⁺ 82 Cu²⁺ 7 Fe³⁺ 93Ni²⁺ 93 Mn²⁺ 120 Al³⁺ 98 EDTA 35

As evident from the results in Table 13, the enzyme activity was greatlyinhibited by Hg²⁺, Cu²⁺, and EDTA. It was also found that the enzyme wasactivated by Ca²⁺ and Mn²⁺.

Amino acid analysis of the N-terminal amino acid sequence of the enzymeby “PROTEIN SEQUENCER MODEL 473A”, an apparatus of Applied Biosystems,Inc., Foster City, USA, revealed that the enzyme had a partial aminoacid sequence of SEQ ID NO:11, i.e.,histidine-valine-serine-alanine-leucine-glycine-asparagine-leucine-leucinein the N-terminal region.

Comparison of the above partial amino acid sequence in the N-terminalregion with that derived from the α-isomaltosylglucosaccharide-formingenzyme from Bacillus globisporus C11 in Experiment 8-1 revealed thatthey had a relatively high homology but differed in the amino acidresidues 1, 4 and 9.

EXPERIMENT 12-2 Property of α-isomaltosylglucosaccharide-forming Enzymeand α-isomaltosyl-transferring Enzyme

A purified specimen of α-isomaltosyl-transferring enzyme, obtained bythe method in Experiment 11-3, was subjected to SDS-PAGE using a 7.5%(w/v) of polyacrylamide gel and then determined for molecular weight bycomparing with the dynamics of standard molecular markerselectrophoresed in parallel, commercialized by Bio-Rad LaboratoriesInc., Brussels, Belgium, revealing that the enzyme had a molecularweight of about 112,000∀20,000 daltons.

A fresh preparation of the above purified specimen was subjected toisoelectrophoresis using a gel containing 2% (w/v) ampholinecommercialized by Amersham Corp., Div. Amersham International, ArlingtonHeights, Ill., USA, and then measured for pHs of protein bands and gelto determine the isoelectric point of the enzyme, revealing that theenzyme had an isoelectric point of about 7.8∀0.5.

The influence of temperature and pH on the activity ofα-isomaltosyl-transferring enzyme was examined in accordance with theassay for the enzyme activity. These results are in FIG. 25 (influenceof temperature) and FIG. 26 (influence of pH). The optimum temperatureof the enzyme was about 50° C. The optimum pH of the enzyme was about6.0 when incubated at 35° C. for 30 min. The thermal stability of theenzyme was determined by incubating the testing enzyme solutions in 20mM acetate buffer (pH 6.0) at prescribed temperatures for 60 min,cooling with water the resulting enzyme solutions, and assaying theremaining enzyme activity of each solution. The pH stability of theenzyme was determined by keeping the testing enzyme solutions in 50 mMbuffers having prescribed pHs at 4° C. for 24 hours, adjusting the pH ofeach solution to 6.0, and assaying the remaining enzyme activity of eachsolution. These results are respectively in FIG. 27 (thermal stability)and FIG. 28 (pH stability). As a result, the enzyme had thermalstability of up to about 45° C. and had pH stability of about 4.5 toabout 10.0.

The influence of metal ions on the activity ofα-isomaltosyl-transferring enzyme was examined in the presence of 1 mMof each metal-ion according to the assay for the enzyme activity. Theresults are in Table 14. TABLE 14 Relative Relative activity activityMetal ion (%) Metal ion (%) None 100 Hg²⁺ 0.5 Zn²⁺ 75 Ba²⁺ 102 Mg²⁺ 95Sr²⁺ 91 Ca²⁺ 100 Pb²⁺ 69 Co²⁺ 92 Fe²⁺ 97 Cu²⁺ 15 Fe³⁺ 90 Ni²⁺ 91 Mn²⁺101 Al³⁺ 94 EDTA 92

As evident from the results in Table 14, the enzyme activity was greatlyinhibited by Hg²⁺ and was also inhibited by Cu²⁺. It was also found thatthe enzyme was not activated by Ca²⁺ and not inhibited by EDTA.

Amino acid analysis of the N-terminal amino acid sequence of the enzymeby “PROTEIN SEQUENCER MODEL 473A”, an apparatus of Applied Biosystems,Inc., Foster City, USA, revealed that the enzyme had a partial aminoacid sequence of SEQ ID NO:3, i.e., isoleucine-asparticacid-glycine-valine-tyrosine-histidine-alanine-proline-tyrosine-glycineat the N-terminal region.

Comparison of the above partial amino acid sequence at the N-terminalregion with that derived from the α-isomaltosylglucosaccharide-formingenzyme from Bacillus globisporus C9 in Experiment 8-2 revealed that theyhad a common amino acid sequence of isoleucine-asparticacid-glycine-valine-tyrosine-histidine-alanine-proline, as shown in SEQID NO:4 at their N-terminal regions.

EXPERIMENT 13 Internal Amino Acid Sequence ofα-isomaltosylglucosaccharide-forming Enzyme andα-isomaltosyl-transferring Enzyme Experiment 13-1 Internal Amino AcidSequence of α-isomaltosylglucosaccharide-forming Enzyme

A part of a purified specimen of α-isomaltosylglucosaccharide-formingenzyme, obtained by the method in Experiment 11-2, was dialyzed against10 mM Tris-HCl buffer (pH 9.0), and the dialyzed solution was dilutedwith a fresh preparation of the same buffer to give a concentration ofabout one milligram per milliliter. One milliliter of the dilute as atest sample was admixed with 20 Φg of “Lysyl Endopeptidase”commercialized by Wako Pure Chemical Industries, Ltd., Tokyo, Japan, andallowed to react at 30° C. for 24 hours to form peptides. The resultantmixtures were subjected to reverse-phase HPLC to separate the peptidesusing “Φ-Bondasphere C18 column” having a diameter of 3.9 mm and alength of 150 mm, a product of Waters Chromatography Div., MILLIPORECorp., Milford, USA, at a flow rate of 0.9 ml/min and at ambienttemperature, and using a liner gradient of acetonitrile increasing from8% (v/v) to 36% (v/v) in 0.1% (v/v) trifluoroacetate over 120 min. Thepeptides eluted from the column were detected by monitoring theabsorbency at a wavelength of 210 nm. Three peptide specimens named PN59with a retention time of about 59 min, PN67 with a retention time ofabout 67 min, and PN87 with a retention time of about 87 min, which hadbeen well separated from other peptides, were separately collected anddried in vacuo and then dissolved in 200 Φl of a solution of 0.1% (v/v)trifluoroacetate and 50% (v/v) acetonitrile. Each peptide specimen wassubjected to a protein sequencer for analyzing amino acid sequence up toeight amino acid residues to obtain amino acid sequences of SEQ IDNOs:12 to 14. The analyzed internal partial amino acid sequences are inTable 15. TABLE 15 Peptide name Internal partial amino acid sequencePN59 aspartic acid-phenylalanine- SEQ ID NO: 12 serine-asparagine-asparagine-proline-threonine-valine PN67tyrosine-threonine-valine-asparagine- SEQ ID NO: 13alanine-proline-alanine-alanine PN87 tyrosine-glutamic acid- SEQ ID NO:14 alanine-glutamic acid- serine-alanine-glutamic acid-leucine

EXPERIMENT 13-2 Internal Amino Acid Sequence ofα-isomaltosyl-transferring Enzyme

A part of a purified specimen of α-isomaltosyl-transferring enzyme,obtained by the method in Experiment 11-3, was dialyzed against 10 mMTris-HCl buffer (pH 9.0), and the dialyzed solution was diluted with afresh preparation of the same buffer to give a concentration of aboutone milligram per milliliter. One milliliter of the dilute as a testsample was admixed with 20 Φg of “Lysyl Endopeptidase” commercialized byWako Pure Chemical Industries, Ltd., Tokyo, Japan, and allowed to reactat 30° C. for 24 hours to form peptides. The resultant mixtures weresubjected to reverse-phase HPLC to separate the peptides using“Φ-Bondasphere C18 column” having a diameter of 3.9 mm and a length of150 mm, a product of Waters Chromatography Div., MILLIPORE Corp.,Milford, USA, at a flow rate of 0.9 ml/min and at ambient temperature,and using a liner gradient of acetonitrile increasing from 4% (v/v) to42.4% (v/v) in 0.1% (v/v) trifluoroacetate over 90 min. The peptideseluted from the column were detected by monitoring the absorbency at awavelength of 210 nm. Three peptide specimens named PN21 with aretention time of about 21 min, PN38 with a retention time of about 38min, and PN69 with a retention time of about 69 min, which had been wellseparated from other peptides, were separately collected and dried invacuo and then dissolved in 200 Φl of a solution of 0.1% (v/v)trifluoroacetate and 50% (v/v) acetonitrile. Each peptide specimen wassubjected to a protein sequencer for analyzing amino acid sequence up toeight amino acid residues, but up to six amino acids residues for PN21,to obtain amino acid sequences of SEQ ID NOs: 15 to 17. The analyzedinternal partial amino acid sequences are in Table 16. TABLE 16 Peptidename Internal partial amino acid sequence PN21asparagine-tryptophan-tryptophan- SEQ ID NO: 15 methionine-serine-lysinePN38 threonine-aspartic acid-glycine-glycine- SEQ ID NO: 16 glutamicacid-methionine-valine-tryptophan PN69asparagine-isoleucine-tyrosine-leucine- SEQ ID NO: 17proline-glutamine-glycine-aspartic acid

EXPERIMENT 14 Production of α-isomaltosylglucosaccharide-forming Enzymefrom Arthrobacter globiformis A19 Strain A19

A liquid nutrient culture medium, consisting of 4.0% (w/v) of “PINE-DEX#4”, a partial starch hydrolysate, 1.8% (w/v) of “ASAHIMEAST”, a yeastextract, 0.1% (w/v) of dipotassium phosphate, 0.06% (w/v) of sodiumphosphate dodecahydrate, 0.05% (w/v) magnesium sulfate heptahydrate, andwater was placed in 500-ml Erlenmeyer flasks in a volume of 100 ml each,autoclaved at 121° C. for 20 minutes to effect sterilization, cooled,inoculated with a stock culture of Arthrobacter globiformis A19, FERMBP-7590, and incubated at 27° C. for 48 hours under rotary shakingconditions of 230 rpm for use as a seed culture.

About 20 L of a fresh preparation of the same nutrient culture medium asused in the above culture were placed in a 30-L fermentor, sterilized byheating, cooled to 27° C., inoculated with 1% (v/v) of the seed culture,and incubated for about 48 hours while stirring under aeration agitationconditions at 27° C. and pH 6.0-9.0. The resultant culture, having about1.1 units/ml of α-isomaltosylglucosaccharide-forming enzyme activity,about 1.7 units/ml of α-isomaltosyl-transferring enzyme activity, andabout 0.35 unit/ml of cyclotetrasaccharide-forming enzyme activity, wascentrifuged at 10,000 rpm for 30 min to obtain about 18 L of asupernatant. Measurement of the supernatant revealed that it had about1.06 units/ml of α-isomaltosylglucosaccharide-forming enzyme activity,i.e., a total enzyme activity of about 19,100 units; about 1.6 units/mlof α-isomaltosyl-transferring enzyme activity, i.e., a total enzymeactivity of about 28,800 units; and about 0.27 unit/ml ofcyclotetrasaccharide-forming enzyme activity, i.e., a total enzymeactivity of about 4,860 units.

The activity of the α-isomaltosylglucosaccharide-forming enzyme fromArthrobacter globiformis A19 was similarly assayed as the method inExperiment 3 except for using 100 mM glycine-NaOH buffer (pH 8.4) wasused as a buffer for substrate.

EXPERIMENT 15 Preparation of Enzyme from Arthrobacter globiformis A19

About 18 L of the supernatant, obtained in Experiment 14, was salted outwith a 60% saturated ammonium sulfate solution and allowed to stand at4° C. for 24 hours. Then, the salted out sediments were collected bycentrifugation at 10,000 for 30 min, dissolved in 10 mM phosphate buffer(pH 7.0), dialyzed against a fresh preparation of the same buffer toobtain about 850 ml of a crude enzyme solution. The crude enzymesolution was revealed to have 8,210 units of theα-isomaltosylglucosaccharide-forming enzyme, about 15,700 units ofα-isomaltosyl-transferring enzyme, and about 20,090 units ofcyclotetrasaccharide-forming enzyme, followed by subjecting it toion-exchange chromatography using 380 ml of “DEAE-TOYOPEARL 650S” gel.When eluted with a linear gradient increasing from 0 M to 0.5 M NaCl,the above enzyme and α-isomaltosyl-transferring enzyme were separatelyeluted from the gel, the former was eluted at a concentration of about0.2 M NaCl, while the latter was eluted at a concentration of about 0.3M NaCl. Under these conditions, fractions with theα-isomaltosylglucosaccharide-forming enzyme activity of the presentinvention and those with α-isomaltosyl-transferring enzyme wereseparately fractionated and collected. Since the facts that nocyclotetrasaccharide-forming activity was found in any fraction in thiscolumn chromatography, and an enzyme solution, obtained by mixingfractions of α-isomaltosylglucosaccharide-forming enzyme and ofα-isomaltosyl-transferring enzyme, showed cyclotetrasaccharide-formingactivity, it was revealed that the activity of formingcyclotetrasaccharide from partial starch hydrolyzates is exerted by thecoaction of the α-isomaltosylglucosaccharide-forming enzyme of thepresent invention and α-isomaltosyl-transferring enzyme.

The following experiments describe a method of separately purifying theα-isomaltosylglucosaccharide-forming enzyme of the present invention andα-isomaltosyl-transferring enzyme:

EXPERIMENT 15-2 Purification of α-isomaltosylglucosaccharide-formingEnzyme

The above fractions with the α-isomaltosylglucosaccharide-forming enzymeof the present invention were pooled and then dialyzed against 10 mMphosphate buffer (pH 7.0) containing 1 M ammonium sulfate, and thedialyzed solution was centrifuged to remove impurities and fed toaffinity chromatography using 500 ml of “SEPHACRYL HR S-200” gel. Theenzyme was adsorbed on the gel and then eluted therefrom with a lineargradient decreasing from 1 M to 0 M ammonium sulfate. As a result, theα-isomaltosylglucosaccharide-forming enzyme adsorbed on the gel waseluted therefrom at a concentration of about 0.2 M ammonium sulfate,followed by collecting fractions with the enzyme activity and poolingthem for use as a final purified specimen. The amount of enzymeactivity, specific activity, and yield of theα-isomaltosylglucosaccharide-forming enzyme in each purification stepare in Table 17. TABLE 17 Specific activity Enzyme* activity of enzyme*Yield Purification step (unit) (unit/mg protein) (%) Culture supernatant19,100 0.11 100 Dialyzed solution after 8,210 0.48 43.0 salting out withammonium sulfate Eluate from ion-exchange 6,890 4.18 36.1 columnchromatography Eluate from affinity 5,220 35.1 27.3 columnchromatographyNote:The symbol “*” means α-isomaltosylglucosaccharide-forming enzyme.

The final purified α-isomaltosylglucosaccharide-forming enzyme specimenwas assayed for purity on gel electrophoresis using a 7.5% (w/v)polyacrylamide gel and detected on the gel as a single protein band,i.e., a high purity enzyme specimen.

EXPERIMENT 15-3 Purification of α-isomaltosyl-transferring Enzyme

Fractions of α-isomaltosyl-transferring enzyme, which had been separatedfrom fractions of α-isomaltosylglucosaccharide-forming enzyme byion-exchange chromatography in Experiment 15-1, were pooled and dialyzedagainst 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate,and the dialyzed solution was centrifuged to remove impurities. Theresulting supernatant was fed affinity column chromatography using 500ml of “SEPHACRYL HR S-200” gel, a gel commercialized by Amersham Corp.,Div. Amersham International, Arlington Heights, Ill., USA. The enzymewas adsorbed on the gel and then eluted with a linear gradientdecreasing from 1 M to 0 M of ammonium sulfate, resulting in an elutionof the enzyme from the gel at a concentration of about 0 M ammoniumsulfate and collecting fractions with the enzyme activity for apartially purified specimen. The amount of enzyme activity, specificactivity, and yield of the α-isomaltosyl-transferring enzyme in eachpurification step are in Table 18. TABLE 18 Specific activity Enzyme*activity of enzyme* Yield Purification step (unit) (unit/mg protein) (%)Culture supernatant 28,800 0.18 100 Dialyzed solution after 15,700 0.9754.5 salting out with ammonium sulfate Eluate from ion-exchange 7,1304.01 24.8 column chromatography Eluate from affinity 1,800 11.9 6.3column chromatographyNote:The symbol “*” means α-isomaltosyl-transferring enzyme.

The partially-purified α-isomaltosyl-transferring enzyme specimen wasassayed for purity on gel electrophoresis using a 7.5% (w/v)polyacrylamide gel and detected on the gel as a main protein band alongwith three minor protein bands.

EXPERIMENT 16 Property of α-isomaltosylglucosaccharide-forming Enzymeand α-isomaltosyl-transferring Enzyme EXPERIMENT 16-1 Property ofα-isomaltosylglucosaccharide-forming Enzyme

A purified specimen of α-isomaltosylglucosaccharide-forming enzyme,obtained by the method in Experiment 15-2, was subjected to SDS-PAGEusing a 7.5% (w/v) of polyacrylamide gel and then determined formolecular weight by comparing with the dynamics of standard molecularmarkers electrophoresed in parallel, commercialized by Bio-RadLaboratories Inc., Brussels, Belgium, revealing that the enzyme had amolecular weight of about 94,000∀20,000 daltons.

A fresh preparation of the above purified specimen was subjected toisoelectrophoresis using a gel containing 2% (w/v) ampholinecommercialized by Amersham Corp., Div. Amersham International, ArlingtonHeights, Ill., USA, and then measured for pHs of protein bands and gelto determine the isoelectric point of the enzyme, revealing that theenzyme had an isoelectric point of about 4.3∀0.5.

The influence of temperature and pH on the activity ofα-isomaltosylglucosaccharide-forming enzyme was examined in accordancewith the assay for the enzyme activity. The influence of temperature wasdetermined in the presence of or in the absence of 1 mM Ca²⁺. Theseresults are in FIG. 29 (influence of temperature) and FIG. 30 (influenceof pH). The optimum temperature of the enzyme was about 60° C. and about65° C. in the absence of and in the presence of 1 mM Ca²⁺, respectively.The optimum pH of the enzyme was about 8.4 when incubated at 35° C. for60 min. The thermal stability of the enzyme was determined by incubatingthe testing enzyme solutions at prescribed temperatures for 60 min in 20mM glycine-NaOH buffer 2+(pH 8.0) and in the absence of or in thepresence of 1 mM Ca²⁺, cooling with water the resulting enzymesolutions, and assaying the remaining enzyme activity of each solution.The pH stability of the enzyme was determined by keeping the testingenzyme solutions in 50 mM buffers having prescribed pHs at 4° C. for 24hours, adjusting the pH of each solution to 8.0, and assaying theremaining enzyme activity of each solution. These results arerespectively in FIG. 31 (thermal stability) and FIG. 32 (pH stability).As a result, the enzyme had thermal stability of up to about 55° C. andabout 60° C. in the absence of and in the presence of 1 mM Ca²⁺,respectively, and had pH stability of about 5.0 to about 9.0.

The influence of metal ions on the activity ofα-isomaltosyl-transferring enzyme was examined in the presence of 1 mMof each metal-ion according to the assay for the enzyme activity. Theresults are in Table 19. TABLE 19 Metal Relative Relative ion activity(%) Metal ion activity (%) None 100 Hg²⁺ 0 Zn²⁺ 56 Ba²⁺ 99 Mg²⁺ 97 Sr²⁺102 Ca²⁺ 106 Pb²⁺ 43 Co²⁺ 93 Fe²⁺ 36 Cu²⁺ 0 Fe³⁺ 35 Ni²⁺ 46 Mn²⁺ 98 Al³⁺37 EDTA 2

As evident from the results in Table 19, it was revealed that the enzymeactivity was greatly inhibited by Hg²⁺, Cu²⁺ and EDTA.

Amino acid analysis of the N-terminal amino acid sequence of the enzymeby “PROTEIN SEQUENCER MODEL 473A”, an apparatus of Applied Biosystems,Inc., Foster City, USA, revealed that the enzyme had a partial aminoacid sequence of SEQ ID NO:18, i.e.,alanine-proline-leucine-glycine-valine-glutamine-arginine-alanine-glutamine-phenylalanine-glutamine-serine-glycineat the N-terminal region.

EXPERIMENT 16-2 Property of α-isomaltosyl-transferring Enzyme

Using a partially-purified specimen of α-isomaltosyl-transferringenzyme, obtained by the method in Experiment 15-3, the influence oftemperature and pH on the enzyme was examined in accordance with theassay for the enzyme activity. These results are in FIG. 33 (influenceof temperature) and FIG. 34 (influence of pH). The optimum temperatureof the enzyme was about 50° C. when incubated at pH 6.0 for 30 min. Theoptimum pH of the enzyme was about 6.5 when incubated at 35° C. for 30min. The thermal stability of the enzyme was determined by incubatingthe testing enzyme solutions in 20 mM acetate buffer (pH 6.0) atprescribed temperatures for 60 min, cooling with water the resultingenzyme solutions, and assaying the remaining enzyme activity of eachsolution. The pH stability of the enzyme was determined by keeping thetesting enzyme solutions in 50 mM buffers having prescribed pHs at 4° C.for 24 hours, adjusting the pH of each solution to 6.0, and assaying theremaining enzyme activity of each solution. These results arerespectively in FIG. 35 (thermal stability) and FIG. 36 (pH stability).As a result, the enzyme had thermal stability of up to about 45° C. andpH stability of about 4.5 to about 9.0.

EXPERIMENT 17 Production of α-isomaltosyl-transferring Enzyme fromArthrobacter ramosus S1 Strain S1

A liquid nutrient culture medium, consisting of 4.0% (w/v) of “PINE-DEX#4”, a partial starch hydrolysate, 1.8% (w/v) of “ASAHIMEAST”, a yeastextract, 0.1% (w/v) of dipotassium phosphate, 0.06% (w/v) of sodiumphosphate dodecahydrate, 0.05% (w/v) magnesium sulfate heptahydrate, andwater was placed in 500-ml Erlenmeyer flasks in a volume of 100 ml each,autoclaved at 121° C. for 20 minutes to effect sterilization, cooled,inoculated with a stock culture of Arthrobacter ramosus S1, FERMBP-7592, and incubated at 27° C. for 48 hours under rotary shakingconditions of 230 rpm for use as a seed culture. About 20 L of a freshpreparation of the same nutrient culture medium as used in the aboveculture were placed in a 30-L fermentor, sterilized by heating, cooledto 27° C., inoculated with 1% (v/v) of the seed culture, and incubatedfor about 48 hours while stirring under aeration agitation conditions at27° C. and pH 6.0-8.0. The resultant culture, having about 0.45 unit/mlof α-isomaltosyl-transferring enzyme activity, was centrifuged at 10,000rpm for 30 min to obtain about 18 L of a supernatant having about 0.44unit/ml of α-isomaltosyl-transferring enzyme activity and a total enzymeactivity of about 7,920 units.

EXPERIMENT 18 Purification of α-isomaltosyl-transferring Enzyme fromArthrobacter ramosus S1

Eighteen liters of a supernatant obtained in Experiment 17 were saltedout with a 80% (w/v) ammonium sulfate at 4° C. for 24 hours, and theresulting sediments were collected by centrifugation at 10,000 rpm for30 min and dialyzed against 10 mM phosphate buffer (pH 7.0) to obtainabout 380 ml of a crude enzyme solution having 6,000 units ofα-isomaltosyl-transferring enzyme. The crude enzyme solution wasdialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammoniumsulfate, and the dialyzed solution was centrifuged to remove impurities.The resulting supernatant was fed affinity column chromatography using500 ml of “SEPHACRYL HR S-200” gel. The enzyme was adsorbed on the geland then eluted sequentially with a linear gradient decreasing from 1 Mto O M of ammonium sulfate and with a linear gradient increasing from 0%(w/v) to 5% (w/v) maltotetraose, resulting in an elution of the enzymefrom the gel at a concentration of about 2% (w/v) maltotetraose andcollecting fractions with the enzyme activity. The fractions were pooledand dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 Mammonium sulfate, and the dialyzed solution was centrifuged to removeimpurities. The supernatant thus obtained was fed to hydrophobic columnchromatography using 380 ml of “BUTYL-TOYOPEARL 650M” gel. When elutedwith a linear gradient decreasing from 1 M to 0 M ammonium sulfate, theα-isomaltosyl-transferring enzyme adsorbed on the gel was elutedtherefrom at about 0.3 M ammonium sulfate, followed by collectingfractions with the enzyme activity for a purified enzyme specimen. Theamount of enzyme activity, specific activity, and yield of theα-isomaltosylglucosaccharide-forming enzyme in each purification stepare in Table 20. TABLE 20 Specific activity Enzyme* activity of enzyme*Yield Purification step (unit) (unit/mg protein) (%) Culture supernatant7,920 0.47 100 Dialyzed solution after 6,000 3.36 75.8 salting out withammonium sulfate Eluate from affinity 5,270 29.9 66.5 columnchromatography Eluate from hydrophobic 4,430 31.1 55.9 columnchromatographyNote:The symbol “*” means α-isomaltosyl-transferring enzyme.

The purified α-isomaltosyl-transferring enzyme specimen in thisexperiment was assayed for purity on gel electrophoresis using a 7.5%(w/v) polyacrylamide gel and detected on the gel as a single proteinband, i.e., a high purity enzyme specimen.

EXPERIMENT 19 Property of α-isomaltosyl-transferring Enzyme

A purified specimen of α-isomaltosyl-transferring enzyme, obtained bythe method in Experiment 18, was subjected to SDS-PAGE using a 7.5%(w/v) of polyacrylamide gel and then determined for molecular weight bycomparing with the dynamics of standard molecular markerselectrophoresed in parallel, commercialized by Bio-Rad LaboratoriesInc., Brussels, Belgium, revealing that the enzyme had a molecularweight of about 116,000∀20,000 daltons.

A fresh preparation of the above purified specimen was subjected toisoelectrophoresis using a gel containing 2% (w/v) ampholinecommercialized by Amersham Corp., Div. Amersham International, ArlingtonHeights, Ill., USA, and then measured for pHs of protein bands and gelto determine the isoelectric point of the enzyme, revealing that theenzyme had an isoelectric point of about 4.2∀0.5.

The influence of temperature and pH on the activity ofα-isomaltosyl-transferring enzyme was examined in accordance with theassay for the enzyme activity. These results are in FIG. 37 (influenceof temperature) and FIG. 38 (influence of pH). The optimum temperatureof the enzyme was about 50° C. when incubated at pH 6.0 for 30 min. Theoptimum pH of the enzyme was about 6.0 when incubated at 35° C. for 30min. The thermal stability of the enzyme was determined by incubatingthe testing enzyme solutions at prescribed temperatures for 60 min in 20mM acetate buffer (pH 6.0), cooling with water the resulting enzymesolutions, and assaying the remaining enzyme activity of each solution.The pH stability of the enzyme was determined by keeping the testingenzyme solutions in 50 mM buffers having prescribed pHs at 4° C. for 24hours, adjusting the pH of each solution to 6.0, and assaying theremaining enzyme activity of each solution. These results arerespectively in FIG. 39 (thermal stability) and FIG. 40 (pH stability).As a result, the enzyme had thermal stability of up to about 45° C. andhad pH stability of about 3.6 to about 9.0.

The influence of metal ions on the activity ofα-isomaltosyl-transferring enzyme was examined in the presence of 1 mMof each metal-ion according to the assay for the enzyme activity. Theresults are in Table 21. TABLE 21 Relative activity Relative activityMetal ion (%) Metal ion (%) None 100 Hg²⁺ 0.1 Zn²⁺ 78 Ba²⁺ 97 Mg²⁺ 99Sr²⁺ 101 Ca²⁺ 103 Pb²⁺ 85 Co²⁺ 91 Fe²⁺ 105 Cu²⁺ 2 Fe³⁺ 75 Ni²⁺ 87 Mn²⁺98 Al³⁺ 93 EDTA 91

As evident from the results in Table 21, it was revealed that the enzymeactivity was greatly inhibited by Hg²⁺ and was also inhibited by Cu²⁺.

Amino acid analysis of the N-terminal amino acid sequence of the enzymeby “PROTEIN SEQUENCER MODEL 473A”, an apparatus of Applied Biosystems,Inc., Foster City, USA, revealed that the enzyme had a partial aminoacid sequence of SEQ ID NO:19, i.e., asparticacid-threonine-leucine-serine-glycine-valine-phenylalanine-histidine-glycine-prolineat the N-terminal region.

EXPERIMENT 20 Action on Saccharides

It was tested whether saccharides can be used as substrates for theα-isomaltosylglucosaccharide-forming enzyme. For the purpose, a solutionof maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose,maltoheptaose, isomaltose, isomaltotriose, panose, isopanose, trehalose,kojibiose, nigerose, neotrehalose, cellobiose, gentibiose, maltitol,maltotriitol, lactose, sucrose, erlose, selaginose, maltosyl glucoside,or isomaltosyl glucoside was prepared.

To each of the above solutions was added two units/g substrate of apurified specimen of α-isomaltosylglucosaccharide-forming enzyme fromeither Bacillus globisporus C9 obtained by the method in Experiment 4-2,Bacillus globisporus C11 obtained by the method in Experiment 7-2,Bacillus globisporus N75 obtained by the method in Experiment 11-2, orArthrobacter globiformis A19 obtained by the method in Experiment 15-2,and the resulting each solution was adjusted to give a substrateconcentration of 2% (w/v) and incubated at 30° C. and pH 6.0, except forusing pH 8.4 for the enzyme from Arthrobacter globiformis A19, for 24hours. The enzyme solutions before and after the enzymatic reactionswere respectively analyzed on TLC disclosed in Experiment 1 to confirmwhether the enzymes acted on these substrates. The results are in Table22. TABLE 22 Enzymatic action Enzyme of Enzyme of Enzyme of Enzyme ofSubstrate Strain C9 Strain C11 Strain N75 Strain A19 Maltose + + + +Maltotriose ++ ++ ++ ++ Maltotetraose +++ +++ +++ +++ Maltopentaose ++++++ +++ +++ Maltohexaose +++ +++ +++ +++ Maltoheptaose +++ +++ +++ +++Isomaltose − − − − Isomaltotriose − − − − Panose − − − − Isopanose ++ ++++ ++ Trehalose − − − − Kojibiose + + + + Nigerose + + + +Neotrehalose + + + + Cellobiose − − − − Gentibiose − − − − Maltitol − −− − Maltotriitol + + + + Lactose − − − − Sucrose − − − − Erlose + + + +Selaginose − − − − Maltosyl glucoside ++ ++ ++ ++ Isomaltosyl glucoside− − − −Note:Before and after the enzymatic reaction, the symbols “−”, “+”, “++”, and“+++” mean that it showed no change, it showed a slight reduction of thecolor spot of the substrate and the formation of other reaction product,it showed a high reduction of the color spot of the substrate and theformation of other reaction product, and it showed a substantialdisappearance of the substrate spot and the# formation of other reaction product, respectively.

As evident from the Table 22, it was revealed that theα-isomaltosylglucosaccharide-forming enzyme of the present inventionwell acted on saccharides having a glucose polymerization degree of atleast three and having a maltose structure at their non-reducing ends,among the saccharides tested. It was also found that the enzyme slightlyacted on saccharides, having a glucose polymerization degree of two,such as maltose, kojibiose, nigerose, neotrehalose, maltotriitol, anderlose.

EXPERIMENT 21 Reaction Product from Maltooligosaccharide EXPERIMENT 21-1Preparation of Reaction Product

To an aqueous solution containing one percent (w/v) of maltose,maltotriose, maltotetraose, or maltopentaose as a substrate was added apurified specimen of α-isomaltosylglucosaccharide-forming enzymeobtained by the method in Experiment 7-2 in an amount of two units/gsolid for the aqueous solutions of maltose and maltotriose, 0.2 unit/gsolid for maltotetraose, and 0.1 unit/g solid for maltopentaose,followed by incubation at 35° C. and pH 6.0 for eight hours. After a10-min incubation at 100° C., the enzymatic reaction was suspended. Theresulting reaction solutions were respectively measured for saccharidecomposition on HPLC using “YMC PACK ODS-AQ303”, a column commercializedby YMC Co., Ltd., Tokyo, Japan, at a column temperature of 40° C. and aflow rate of 0.5 ml/min of water, and using as a detector “RI-8012”, adifferential refractometer commercialized by Tosoh Corporation, Tokyo,Japan. The results are in Table 23. TABLE 23 Substrate Saccharide Malto-as reaction produc Maltose Maltotriose tetraose Maltopentaose Glucose8.5 0.1 0.0 0.0 Maltose 78.0 17.9 0.3 0.0 Maltotriose 0.8 45.3 22.7 1.9Maltotetraose 0.0 1.8 35.1 19.2 Maltopentaose 0.0 0.0 3.5 34.4Maltohexaose 0.0 0.0 0.0 4.6 Isomaltose 0.5 0.0 0.0 0.0 Glucosylmaltose8.2 1.2 0.0 0.0 Glucosylmaltotriose 2.4 31.5 6.8 0.0 X 0.0 2.1 30.0 11.4Y 0.0 0.0 1.4 26.8 Z 0.0 0.0 0.0 1.7 Others 0.6 0.1 0.2 0.0Note:In the table, glucosylmaltose means α-isomaltosylglucose alias6²-O-α-glucosylmaltose or panose; glucosylmaltotriose meansα-isomaltosylglucose alias 6³-O-α-glucosylmaltotriose; X means theα-isomaltosylmaltotriose in Experiment 11-2, alias6⁴-O-α-glucomaltotetraose; Y means the α-isomaltosylmaltotetraose inExperiment 11-2, alias 6⁵-O-α-glucosylmaltopentaose;# and Z means an unidentified saccharide.

As evident from the results in Table 23, it was revealed that, after theaction of the enzyme of the present invention, glucose andα-isomaltosylglucose alias 6²-O-α-glucosylmaltose or panose were mainlyformed from maltose as a substrate; and maltose and α-isomaltosylglucosealias 6³-O-α-glucosylmaltotriose were mainly formed along with smallamounts of glucose, maltotetraose, α-isomaltosylglucose alias6²-O-α-glucosylmaltose or panose, and the product X. Also, it wasrevealed that maltotriose and the product X were mainly formed frommaltotetraose as a substrate along with small amounts of maltose,maltopentaose, α-isomaltosylglucose alias 6³-O-α-glucosylmaltotriose;and the product Y; and that maltotetraose and the product Y were mainlyformed from maltopentaose as a substrate along with small amounts ofmaltotriose, maltohexaose, and the products X and Z.

The product X as a main product from maltotetraose as a substrate andthe product Y as a main product from maltopentaose as a substrate wererespectively isolated and purified as follows: The products X and Y wererespectively purified on HPLC using “YMC PACK ODS-A R355-15S-15 12A”, aseparatory HPLC column commercialized by YMC Co., Ltd., Tokyo, Japan, toisolate a specimen of the product X having a purity of at least 99.9%from the reaction product from maltotetraose in a yield of about 8.3%,d.s.b., and a specimen of the product Y having a purity of at least99.9% from the reaction product from maltotetraose in a yield of about11.5%, d.s.b.

EXPERIMENT 21-2 Structural Analysis on Reaction Product

Using the products X and Y obtained by the method in Experiment 21-1,they were subjected to methyl analysis and NMR analysis in a usualmanner. The results on their methyl analyses are in Table 24. For theresults on their NMR analyses, FIG. 41 is a ¹H-NMR spectrum for theproduct X and FIG. 42 is for the product Y. The ¹³C-NMR spectra for theproducts X and Y are respectively FIGS. 43 and 44. The assignment of theproducts X and Y are tabulated in Table 25. TABLE 24 Analyzed Ratiomethyl compound Product X Product Y 2,3,4-trimethyl compound 1.00 1.002,3,6-trimethyl compound 3.05 3.98 2,3,4,6-tetramethyl compound 0.820.85

Based on these results, the product X formed from maltotetraose via theaction of the α-isomaltosylglucosaccharide-forming enzyme of the presentinvention was revealed as a pentasaccharide, in which a glucose residuebinds via the α-linkage to OH-6 of glucose at the non-reducing end ofmaltotetraose, i.e., α-isomaltosylmaltotriose alias6⁴-O-α-glucosylmaltotetraose, represented by Formula 1.α-D-Glcp-(166)-α-D-Glcp-(164)-α-D-Glcp-(164)-α-D-Glcp-(164)-D-Glcp  Formula1:

The product Y formed from maltopentaose was revealed as ahexasaccharide, in which a glucosyl residue binds via the α-linkage toOH-6 of glucose at the non-reducing end of maltopentaose, i.e.,α-isomaltosylmaltotetraose alias 6⁵-O-α-glucosylmaltopentaose,represented by Formula 2.α-D-Glcp-(166)-α-D-Glcp-(164)-α-D-Glcp-(164)-α-D-Glcp-(164)-α-D-Glcp-(164)-D-Glcp  Formula2: TABLE 25 Glucose Carbon Chemical shift on NMR (ppm) number numberProduct X Product Y a 1a 100.8 100.8 2a 74.2 74.2 3a 75.8 75.7 4a 72.272.2 5a 74.5 74.5 6a 63.2 63.1 b 1b 102.6 102.6 2b 74.2 74.2 3b 75.875.7 4b 72.1 72.1 5b 74.0 74.0 6b 68.6 68.6 c 1c 102.3 102.3 2c 74.274.2 3c 76.0 76.0 4c 79.6 79.5 5c 73.9 73.9 6c 63.2 63.1 d 1d 102.2102.3 2d 74.0 (α), 74.4 (β) 74.2 3d 76.0 76.0 4d 79.8 79.5 5d 73.9 73.96d 63.2 63.1 e 1e 94.6 (α), 98.5 (β) 102.1 2e 74.2 (α), 76.7 (β) 74.0(α), 74.4 (β) 3e 75.9 (α), 78.9 (β) 76.0 4e 79.6 (α), 79.4 (β) 79.8 5e72.6 (α), 77.2 (β) 73.9 6e 63.4 (α), 63.4 (β) 63.1 f 1f 94.6 (α), 98.5(β) 2f 74.2 (α), 76.7 (β) 3f 76.0 (α), 78.9 (β) 4f 79.6 (α), 79.5 (β) 5f72.6 (α), 77.2 (β) 6f 63.3 (α), 63.3 (β)

Based on these results, it was concluded that theα-isomaltosylglucosaccharide-forming enzyme of the present inventionacts on maltooligosaccharides as shown below:

(1) The enzyme acts on as a substrate maltooligosaccharides having aglucose polymerization degree of at least two linked via the α-1,4linkage, and catalyzes the intermolecular 6-glucosyl-transferringreaction in such a manner of transferring a glucosyl residue at thenon-reducing end of a maltooligosaccharide molecule to C-6 of thenon-reducing end of other maltooligosaccharide molecule to form both anα-isomaltosylglucosaccharide alias 6-O-α-glucosylmaltooligosaccharide,having a 6-O-α-glucosyl residue and a higher glucose polymerizationdegree by one as compared with the intact substrate, and amaltooligosaccharide with a reduced glucose polymerization by one ascompared with the intact substrate; and

(2) The enzyme slightly catalyzes the 4-glucosyl-transferring reactionand forms both a maltooligosaccharide, having an increased glucosepolymerization by one as compared with the intact substrate, and amaltooligosaccharide having a reduced glucose polymerization degree byone as compared with the intact substrate.

EXPERIMENT 22 Test on Reducing-Power Formation

The following test was carried out to study whether theα-isomaltosylglucosaccharide-formation enzyme of the present inventionhad the reducing power. To a 1% (w/v) aqueous solution of maltotetraoseas a substrate was added 0.25 unit/g substrate, d.s.b., of either ofpurified specimens of α-isomaltosylglucosaccharide-forming enzyme fromBacillus globisporus C9 obtained by the method in Experiment 4-2,Bacillus globisporus C11 obtained by the method in Experiment 7-2,Bacillus globisporus N75 obtained by the method in Experiment 11-2, orArthrobacter globiformis A19 obtained by the method in Experiment 15-2,and incubated at 35° C. and pH 6.0, except that pH 8.4 was used for theenzyme from Arthrobacter globiformis A19. During enzymatic reaction, aportion of each reaction solution was sampled at prescribed timeintervals and measured for reducing power after keeping the sampledsolutions at 100° C. for 10 min to suspend the enzymatic reaction.Before and after the enzymatic reaction, the reducing saccharide contentand the total sugar content were respectively quantified by theSomogyi-Nelson's method and the anthrone-sulfuric acid reaction method.The percentage of forming reducing power was calculated by the followingequation:Equation: $\begin{matrix}{{Percentage}\quad{of}\quad{forming}} \\{{reducing}\quad{power}\quad(\%)}\end{matrix} = {{{\frac{A\quad R}{A\quad T} - \frac{B\quad R}{B\quad T}}} \times 100}$AR: Reducing sugar content after enzymatic reaction.AT: Total sugar content after enzymatic reaction.BR: Reducing sugar content before enzymatic reaction.BT: Total sugar content before enzymatic reaction.

The results are in Table 26. TABLE 26 Percentage of forming Reactionreducing power (%) time Enzyme of Enzyme of Enzyme of Enzyme of (hour)Strain C9 Strain C11 Strain N75 Stain A19 0 0.0 0.0 0.0 0.0 1 0.0 0.10.1 0.0 2 0.1 0.0 0.0 0.1 4 0.1 0.1 0.0 0.0 8 0.0 0.0 0.1 0.1

As evident from the results in Table 26, it was revealed that theα-isomaltosylglucosaccharide-forming enzyme of the present invention didnot substantially increase the reducing power of the reaction productwhen acted on maltotetraose as a substrate; the enzyme did not havehydrolyzing activity or had only an undetectable level of such activity.

EXPERIMENT 23 Test on Dextran Formation

To study whether the α-isomaltosylglucosaccharide-formation enzyme ofthe present invention has the ability of to form dextran, it was testedin accordance with the method in Bioscience Biotechnology andBiochemistry, Vol. 56, pp. 169-173 (1992). To a 1% (w/v) aqueoussolution of maltotetraose as a substrate was added 0.25 unit/gsubstrate, d.s.b., of either of purified specimens ofα-isomaltosylglucosaccharide-forming enzyme from Bacillus globisporus C9obtained by the method in Experiment 4-2, Bacillus globisporus C11obtained by the method in Experiment 7-2, Bacillus globisporus N75obtained by the method in Experiment 11-2, or Arthrobacter globiformisA19 obtained by the method in Experiment 15-2, and incubated at 35° C.and pH 6.0, except that pH 8.4 was used for the enzyme from Arthrobacterglobiformis A19, for four or eight hours. After completion of theenzymatic reaction, the reaction was suspended by heating at 100° C. for15 min. Fifty microliters of each of the reaction mixtures were placedin a centrifugation tube and then admixed and sufficiently stirred with3-fold volumes of ethanol, followed by standing at 4° C. for 30 min.Thereafter, each mixture solution was centrifuged at 15,000 rpm for fiveminutes and, after removing supernatant, the resulting sediment wasadmixed with one milliliter of 75% (w/w) ethanol solution and stirredfor washing. Each resulting solution was centrifuged to removesupernatant, dried in vacuo, and then admixed and sufficiently stirredwith one milliliter of deionized water. The total sugar content, interms of glucose, of each resulting solution was quantified by thephenol-sulfuric acid method. As a control, the total sugar content wasdetermined similarly as in the above except for using either of purifiedspecimens of α-isomaltosylglucosaccharide-forming enzyme from Bacillusglobisporus C9, Bacillus globisporus C11, Bacillus globisporus N75, andArthrobacter globiformis A19, which had been inactivated at 100° C. for10 min. The content of dextran formed was calculated by the followingequation:Content of dextran formed (mg/ml)=[(Total sugar content for testsample)]−[(Total sugar content for control sample)]×20  Equation:

The results are in Table 27. TABLE 27 Reaction Content of dextran formed(mg/ml) time Enzyme of Enzyme of Enzyme of Enzyme of (hour) Strain C9Strain C11 Strain N75 Stain A19 4 0.0 0.0 0.0 0.0 8 0.0 0.0 0.0 0.0

As evident from the results in Table 27, it was revealed that theα-isomaltosylglucosaccharide-forming enzyme of the present invention didnot substantially have the action of forming dextran or had only anundetectable level of such activity because it did not form dextran whenit acted on maltotetraose.

EXPERIMENT 24 Transfer-Acceptor Specificity

Using different saccharides, it was tested whether the saccharides wereused as transferring-acceptors for theα-isomaltosylglucosaccharide-forming enzyme of the present invention. Asolution of D-glucose, D-xylose, L-xylose, D-galactose, D-fructose,D-mannose, D-arabinose, D-fucose, D-psicose, L-sorbose, L-rhamnose,methyl-α-glucopyranoside, methyl-β-glucopyranoside,N-acetyl-glucosamine, sorbitol, trehalose, isomaltose, isomaltotriose,cellobiose, gentibiose, glycerol, maltitol, lactose, sucrose,α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or L-ascorbic acid wasprepared.

To each solution with a saccharide concentration of 1.6% was added“PINE-DEX #100”, a partial starch hydrolysate, as a saccharide donor, togive a concentration of 4%, and admixed with one unit/g saccharidedonor, d.s.b., of either of purified specimens ofα-isomaltosylglucosaccharide-forming enzyme from Bacillus globisporus C9obtained by the method in Experiment 4-2, Bacillus globisporus C11obtained by the method in Experiment 7-2, Bacillus globisporus N75obtained by the method in Experiment 11-2, or Arthrobacter globiformisA19 obtained by the method in Experiment 15-2, and incubated at 30° C.and pH 6.0, except that pH 8.4 was used for the enzyme from Arthrobacterglobiformis A19, for 24 hours. The reaction mixtures of thepost-enzymatic reactions were analyzed on gas chromatography(abbreviated as “GLC” hereinafter) for monosaccharides and disaccharidesas acceptors, and on HPLC for trisaccharides as acceptors to confirmwhether these saccharides could be used as their transfer acceptors. Inthe case of performing GLC, the following apparatuses and conditions areused: GLC apparatus, “GC-16A” commercialized by Shimadzu Corporation,Tokyo, Japan; column, a stainless-steel column, 3 mm in diameter and 2 min length, packed with 2% “SILICONE OV-17/CHROMOSOLV W”, commercializedby GL Sciences Inc., Tokyo, Japan; carrier gas, nitrogen gas at a flowrate of 40 ml/min under temperature conditions of increasing from 160°C. to 320° C. at an increasing temperature rate of 7.5° C./min; anddetection, a hydrogen flame ionization detector. In the case of HPLCanalysis, the apparatuses and conditions used are: HPLC apparatus,“CCPD” commercialized by Tosoh Corporation, Tokyo, Japan; column,“ODS-AQ-303” commercialized by YMC Co., Ltd., Tokyo, Japan; eluent,water at a flow rate of 0.5 ml/min; and detection, a differentialrefractometer. The results are in Table 28. TABLE 28 Product oftransferring reaction Enzyme of Enzyme of Enzyme of Enzyme of SaccharideStrain C9 Strain C11 Strain N75 Stain A19 D-Glucose + + + + D-Xylose ++++ ++ + L-Xylose ++ ++ ++ + D-Galactose + + + ∀ D-Fructose + + + +D-Mannose − − − ∀ D-Arabinose ∀ ∀ ∀ ∀ D-Fucose + + + ∀ D-Psicose + + + +L-Sorbose + + + + L-Rhamnose − − − − Methyl-α- ++ ++ ++ ++glucopyranoside Methyl-β- ++ ++ ++ ++ glucopyranosideN-Acetyl-glucosamine + + + − Sorbitol − − − − Trehalose ++ ++ ++ ++Isomaltose ++ ++ ++ + Isomaltotriose ++ ++ ++ ∀ Cellobiose ++ ++ ++ ++Gentibiose ++ ++ ++ + Glycerol + + + + Maltitol ++ ++ ++ ++ Lactose ++++ ++ ++ Sucrose ++ ++ ++ ++ α-Cyclodextrin − − − − β-Cyclodextrin − − −− γ-Cyclodextrin − − − − L-Ascorbic acid + + + +Note:In the table, the symbols “−”, “∀”, “+”, and “++” mean that nosaccharide-transferred product was detected through transfer reaction toacceptor; a saccharide-transferred product was detected in an amountless than one percent through transfer reaction to acceptor; asaccharide-transferred product was detected in an amount over onepercent but less than ten percent throughtransfer reaction to acceptor;and a saccharide-transferred product# was detected in an amount over ten percent through transfer reactionto acceptor.

As evident from the results in Table 28, it was revealed that theα-isomaltosylglucosaccharide of the present invention utilizes differenttypes of saccharides as transfer acceptors; theα-isomaltosylglucosaccharide-forming enzyme from Stains C9, C11 and N75advantageously transfer, particularly, to D-/L-xylose,methyl-α-glucopyranoside, methyl-β-glucopyranoside, trehalose,isomaltose, isomaltotriose, cellobiose, gentibiose, maltitol, lactose,and sucrose; then transfer to D-glucose, D-fructose, D-fucose,D-psicose, L-sorbose, N-acetylglucosamine, glycerol, and L-ascorbicacid; and further to D-arabinose. Theα-isomaltosylglucosaccharide-forming enzyme from Strain A19 welltransfers, particularly, to methyl-α-glucopyranoside,methyl-β-glucopyranoside, trehalose, cellobiose, maltitol, lactose, andsucrose; then transfers to D-glucose, D-/L-xylose, D-fructose,D-psicose, L-sorbose, isomaltose, gentibiose, glycerol, and L-ascorbicacid; and further to D-galactose, D-mannose, D-arabinose, D-fucose, andisomaltotriose.

The properties of the α-isomaltosylglucosaccharide of the presentinvention described above were compared with those of a previouslyreported enzyme having 6-glucosyl-transferring action; a dextrindextranase disclosed in “Bioscience Biotechnology and Biochemistry, Vol.56, pp. 169-173 (1992); and a transglucosidase disclosed in “NipponNogeikagaku Kaishi”, Vol. 37, pp. 668-672 (1963). The results are inTable 29. TABLE 29 α-Isomaltosyl-glucosaccharide- forming enzyme of thepresent invention Strain Strain Strain Strain Dextrin dextranaseTransglucosidase Property C9 C11 N75 A19 Control Control HydrolysisNegative Negative Negative Negative Negative Mainly positive activityOptimum pH 6.0-6.5 6.0 6.0 8.4 4.0-4.2 3.5 Inhibition Positive PositivePositive Positive Negative Negative by EDTA

As evident from Table 29, the α-isomaltosylglucosaccharide-formingenzyme of the present invention had outstandingly novel physicochemicalproperties completely different from those of known dextrin dextranaseand transglucosidase.

EXPERIMENT 25 Formation of Cyclotetrasaccharide

The test on the formation of cyclotetrasaccharide by theα-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme was conducted using saccharides. Forthe test, prepared a solution of maltose, maltotriose, maltotetraose,maltopentaose, amylose, soluble starch, “PINE-DEX #100” (a partialstarch hydrolyzate commercialized by Matsutani Chemical Ind., Tokyo,Japan), or glycogen from oyster commercialized by Wako Pure ChemicalIndustries Ltd., Tokyo, Japan was prepared.

To each of these solutions with a respective concentration of 0.5%, oneunit/g solid of a purified specimen ofα-isomaltosylglucosaccharide-forming enzyme from Strain C11 obtained bythe method in Experiment 7-2 and 10 units/g solid of a purified specimenof α-isomaltosyl-transferring enzyme from Strain C11 obtained by themethod in Experiment 7-3, and the resulting mixture was subjected to anenzymatic reaction at 30° C. and pH 6.0. The enzymatic conditions werethe following four systems:

(1) After the α-isomaltosylglucosaccharide-forming enzyme was allowed toact on a saccharide solution for 24 hours, the enzyme was inactivated byheating, and then the α-isomaltosyl-transferring enzyme was allowed toact on the resulting mixture for 24 hours and inactivated by heating;

(2) After the α-isomaltosylglucosaccharide-forming enzyme and theα-isomaltosyl-transferring enzyme were allowed in combination to act ona saccharide solution for 24 hours, then the saccharides wereinactivated by heating;

(3) After only the α-isomaltosylglucosaccharide-forming enzyme wasallowed to act on a saccharide solution for 24 hours, then the enzymewas inactivated by heating; and

(4). After only the α-isomaltosyl-transferring enzyme was allowed to acton a saccharide solution for 24 hours, then the enzyme was inactivatedby heating.

To determine the formation level of cyclotetrasaccharide in eachreaction mixture after the heating, the reaction mixture was treatedwith a similar α-glucosidase and glucoamylase as in Experiment 1 tohydrolyze the remaining reducing oligosaccharides, followed by thequantitation of cyclotetrasaccharide on HPLC. The results are in Table30. TABLE 30 Formation yield of cyclotetrasaccharide (%) Substrate A B CD Maltose 4.0 4.2 0.0 0.0 Maltotriose 10.2 12.4 0.0 0.0 Maltotetraose11.3 21.5 0.0 0.0 Maltopentaose 10.5 37.8 0.0 0.0 Amylose 3.5 31.6 0.00.0 Soluble starch 5.1 38.2 0.0 0.0 Partial starch 6.8 63.7 0.0 0.0hydrolyzate Glycogen 10.2 86.9 0.0 0.0Note:The symbols “A”, “B”, “C” and “D” mean thatα-isomaltosylglucosaccharide-forming enzyme was first allowed to act ona substrate and then α-isomaltosyl-transferring enzyme was allowed actedon the substrate, the α-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme were allowed to coact on a substrate,only α-isomaltosylglucosaccharide-forming enzyme was allowed to act on asubstrate, and only# α-isomaltosyl-transferring enzyme was allowed to act on a substrate.

As evident from the results in Table 30, no cyclotetrasaccharide wasformed from any of the saccharides tested by the action of onlyα-isomaltosylglucosaccharide-forming enzyme orα-isomaltosyl-transferring enzyme, but cyclotetrasaccharide was formedby the coaction of these enzymes. It was revealed that the formationlevel was relatively low as below about 11% whenα-isomaltosyl-transferring enzyme was allowed to act on the saccharidesafter the action of α-isomaltosylglucosaccharide-forming enzyme, whilethe level was increased by simultaneously allowing the enzymes to act onevery saccharide tested, particularly, increased to about 87% and about64% when allowed to act on glycogen and partial starch hydrolyzate,respectively.

Based on the reaction properties of α-isomaltosylglucosaccharide-formingenzyme and α-isomaltosyl-transferring enzyme, the formation mechanism ofcyclotetrasaccharide by the coaction of these enzymes is estimated asfollows:

(1) The α-isomaltosylglucosaccharide-forming enzyme of the presentinvention acts on a glucose residue at the non-reducing end of an α-1,4glucan chain of glycogen and partial starch hydrolyzates, etc., andintermolecularly transfers the glucose residue to OH-6 of a glucoseresidue at the non-reducing end of other α-1,4 glucan chain of glycogento form an α-1,4 glucan chain having an α-isomaltosyl residue at thenon-reducing end;

(2) α-isomaltosyl-transferring enzyme acts on the α-1,4 glucan chainhaving an α-isomaltosyl residue at the non-reducing end andintermolecularly transfers the isomaltosyl residue to C-3 of glucoseresidue at the non-reducing end of another α-1,4 glucan chain havingisomaltosyl residue at the non-reducing end to form an α-1,4 glucanchain having an isomaltosyl-1,3-isomaltosyl residue at the non-reducingend;

(3) Then, α-isomaltosyl-transferring enzyme acts on the α-1,4 glucanchain having an isomaltosyl-1,3-isomaltosyl residue at the non-reducingend and releases the isomaltosyl-1,3-isomaltosyl residue from the α-1,4glucan chain via the intramolecular transferring reaction to cyclize thereleased isomaltosyl-1,3-isomaltosyl residue into cyclotetrasaccharide;

(4) From the released α-1,4 glucan chain, cyclotetrasaccharide is formedthrough the sequential steps (1) to (3). Thus, it is estimated that thecoaction of α-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme in such a cyclic manner as indicatedabove increases the formation of cyclotetrasaccharide.

EXPERIMENT 26 Influence of Liquefraction Degree of Starch

A 15% corn starch suspension was prepared, admixed with 0.1% calciumcarbonate, adjusted to pH 6.0, and then mixed with 0.2-2.0% per gramstarch of “TERMAMYL 60L”, an α-amylase specimen commercialized by NovoIndutri A/S, Copenhagen, Denmark, followed by the enzymatic reaction at95° C. for 10 min. Thereafter, the reaction mixture was autoclaved at120° C. for 20 min, promptly cooled to about 35° C. to obtain aliquefied starch with a DE (dextrose equivalent) of 3.2-20.5. To theliquefied starch were added two units/g solid of a purified specimen ofα-isomaltosylglucosaccharide-forming enzyme from Strain C11 obtained bythe method in Experiment 7-2, and 20 units/g solid of a purifiedspecimen of α-isomaltosyl-transferring enzyme from Strain C11 obtainedby the method in Experiment 7-3, followed by the incubation at 35° C.for 24 hours. After completion of the reaction, the reaction mixture washeated at 100° C. for 15 min to inactivate the remaining enzymes. Then,the reaction mixture thus obtained was treated with α-glucosidase andglucoamylase similarly as in Experiment 1 to hydrolyze the remainingreducing oligosaccharides, followed by quantifying the formedcyclotetrasaccharide on HPLC. The results are in Table 31. TABLE 31Amount of α-amylase Yield of per starch (%) DE cyclotetrasaccharide (%)0.2 3.2 54.5 0.4 4.8 50.5 0.6 7.8 44.1 1.0 12.5 39.8 1.5 17.3 34.4 2.020.5 30.8

As evident from the results in Table 31, it was revealed that theformation of cyclotetrasaccharide by the coaction ofα-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme is influenced by the liquefractiondegree of starch, i.e., the lower the liquefraction degree or the lowerthe DE the more the yield of cyclotetrasaccharide from starch becomes.On the contrary, the higher the liquefraction degree or the high the DEthe lower the yield of cyclotetrasaccharide from starch becomes. It wasrevealed that a suitable liquefraction degree is a DE of about 20 orlower, preferably, DE of about 12 or lower, more preferably, DE of about5 or lower.

EXPERIMENT 27 Influence of Concentration of Partial Starch Hydrolyzate

Aqueous solutions of “PINE-DEX #100”, a partial starch hydrolyzate witha DE of about 2 to about 5, having a final concentration of 0.5-40%,were prepared and respectively admixed with one unit/g solid of apurified specimen of α-isomaltosylglucosaccharide-forming enzyme fromStrain C11 obtained by the method in Experiment 7-2 and 10 units/g solidof a purified specimen of α-isomaltosyl-transferring enzyme from StrainC11 obtained by the method in Experiment 7-3, followed by the coactionof these enzymes at 30° C. and pH 6.0 for 48 hours. After completion ofthe reaction, the reaction mixture was heated at 100° C. for 15 min toinactivate the remaining enzymes, and then treated with α-glucosidaseand glucoamylase similarly as in Experiment 1 to hydrolyze the remainingreducing oligosaccharides, followed by quantifying the formedcyclotetrasaccharide on HPLC. The results are in Table 32. TABLE 32Concentration of Formation yield of PINE-DEX (%) cyclotetrasaccharide(%) 0.5 63.6 2.5 62.0 5 60.4 10 57.3 15 54.6 20 51.3 30 45.9 40 35.9

As is evident from the results in Table 32, the formation yield ofcyclotetrasaccharide was about 64% at a low concentration of 0.5%, whileit was about 40% at a high concentration of 40%. The fact indicated thatthe formation yield of cyclotetrasaccharide increased depending on theconcentration of partial starch hydrolyzate as a substrate. The resultrevealed that the formation yield of cyclotetrasaccharide increased asthe decrease of partial starch hydrolyzate.

EXPERIMENT 28 Influence of the Addition of CyclodextrinGlucanotransferase

A 15% aqueous solution of “PINE-DEX #100”, a partial starch hydrolyzatewas prepared and admixed with one unit/g solid of a purified specimen ofα-isomaltosylglucosaccharide-forming enzyme from Strain C11 obtained bythe method in Experiment 7-2, 10 units/g solid of a purified specimen ofα-isomaltosyl-transferring enzyme from Strain C11 obtained by the methodin Experiment 7-3, and 0-0.5 unit/g solid of cyclodextringlucanotransferase (CGTase) from a microorganism of the species Bacillusstearothermophilus, followed by the coaction of these enzymes at 30° C.and pH 6.0 for 48 hours. After completion of the reaction, the reactionmixture was heated at 100° C. for 15 min to inactivate the remainingenzymes, and then treated with α-glucosidase and glucoamylase similarlyas in Experiment 1 to hydrolyze the remaining reducing oligosaccharides,followed by quantifying the formed cyclotetrasaccharide on HPLC. Theresults are in Table 33. TABLE 33 Amount of CGTase added Formation yieldof (unit) cyclotetrasaccharide (%) 0 54.6 2.5 60.1 5 63.1 10 65.2

As evident from the Table 33, it was revealed that the addition ofCGTase increased the formation yield of cyclotetrasaccharide.

EXPERIMENT 29 Preparation of Cyclotetrasaccharide

About 100 L of a 4% (w/v) aqueous solution of corn phytoglycogen,commercialized by Q.P. Corporation, Tokyo, Japan, was prepared, adjustedto pH 6.0 and 30° C., and then admixed with one unit/g solid of apurified specimen of α-isomaltosylglucosaccharide-forming enzyme fromStrain C11 obtained by the method in Experiment 7-2, 10 units/g solid ofa purified specimen of α-isomaltosyl-transferring enzyme from Strain C11obtained by the method in Experiment 7-3, followed by the incubation for48 hours. After completion of the reaction, the reaction mixture washeated at 100° C. for 10 min to inactivate the remaining enzymes, and aportion of the reaction mixture was sampled and then quantified on HPLCfor the formation yield of cyclotetrasaccharide, revealing that itcontained about 84% cyclotetrasaccharide, on a saccharide compositionbasis. The reaction mixture was adjusted to pH 5.0 and 45° C., and thentreated with α-glucosidase and glucoamylase similarly as in Experiment 1to hydrolyze the remaining reducing oligosaccharides, etc. The resultingmixture was adjusted to pH 5.8 by the addition of sodium hydroxide andthen incubated at 90° C. for one hour to inactivate the remainingenzymes and filtered to remove insoluble substances. The filtrate wasconcentrated using a reverse osmosis membrane to give a concentration ofabout 16%, d.s.b., and the concentrate was in a usual manner decolored,desalted, filtered, and concentrated to obtain about 6.2 kg of asaccharide solution with a solid content of about 3,700 g.

The saccharide solution was fed to a column packed with about 225 L of“AMBERLITE CR-1310 (Na-form)”, an ion-exchange resin commercialized byJapan Organo Co., Ltd., Tokyo, Japan, and chromatographed at a columntemperature of 60° C. and a flow rate of about 45 L/h. While thesaccharide composition of eluate from the column was monitored by HPLCas described in Experiment 1, fractions of cyclotetrasaccharide with apurity of at least 98% were collected, and in a usual manner desalted,decolored, filtered, and concentrated to obtain about 7.5 kg of asaccharide solution with a solid content of about 2,500 g solids. HPLCmeasurement for saccharide composition of the saccharide solutionrevealed that it contained cyclotetrasaccharide with a purity of about99.5%.

EXPERIMENT 30 Crystallization of Cyclotetrasaccharide in AqueousSolution

A fraction of cyclotetrasaccharide with a purity of at least 98%,obtained by the method in Experiment 29, was concentrated by evaporationto give a concentration of about 50%, d.s.b. About five kilograms of theconcentrate was placed in a cylindrical plastic vessel and thencrystallized to obtain a white crystalline powder by lowering thetemperature of the concentrate from 65° C. to 20° C. over about 20 hoursunder gentle rotatory conditions. FIG. 45 is a microscopic photograph ofsuch cyclotetrasaccharide. The above crystallized concentrate wasseparated by a centrifugal filter to obtain 1,360 g of a crystallineproduct by wet weight, which was then further dried at 60° C. for threehours to obtain 1,170 g of a crystalline powder of cyclotetrasaccharide.HPLC measurement of the crystalline powder revealed that it containedcyclotetrasaccharide with a quite high purity of at least 99.9%. Whenanalyzed on powder x-ray diffraction analysis, the cyclotetrasaccharidein a crystalline powder form had a diffraction spectrum havingcharacteristic main diffraction angles (2θ) of 10.1°, 15.2°, 20.3°, and25.5° in FIG. 46. The Karl Fischer method of the crystalline powderrevealed that it had a moisture content of 13.0%, resulting in a findingthat it was a crystal of cyclotetrasaccharide having five or six molesof water per one mole of the crystal.

The thermogravimetric analysis of the cyclotetrasaccharide in acrystalline form gave a thermogravimetric curve in FIG. 47. Based on therelationship between the weight change and the temperature, it wassuccessively found that the weight reduction corresponding to four orfive moles of water was observed up to a temperature of 150° C., theweight reduction corresponding to one mole of water at around 250° C.,and the weight reduction corresponding to the decomposition ofcyclotetrasaccharide at a temperature of about 280° C. or higher. Theseresults confirmed that the cyclotetrasaccharide crystal, penta- orhexa-hydrate, of the present invention releases four or five moles ofwater to changes into a monohydrate crystal when heated up to 150° C. atnormal pressure, and further releases one mole of water to change intoan anhydrous crystal after being heated up to 250° C.

EXPERIMENT 31 Conversion into Cyclotetrasaccharide, Monohydrate

Cyclotetrasaccharide, penta- or hexa-hydrate, in a crystalline powderform, obtained by the method in Experiment 30, was placed in a glassvessel, and kept in an oil bath, which had been preheated at 140° C.,for 30 min. Unlike quite different from the result from the powder x-raydiffraction analysis of the intact cyclotetrasaccharide, penta- orhexa-hydrate, the powder x-ray analysis of the cyclotetrasaccharidepowder thus obtained gave a characteristic diffraction spectrum havingmain diffraction angles (2θ) of 8.3°, 16.6°, 17.0°, and 18.2° in FIG.48. The Karl Fischer method of the crystalline powder revealed that ithad a moisture content of about 2.7%, resulting in a finding that it wasa crystal of cyclotetrasaccharide having one mole of water per one moleof the crystal. The thermogravimetric analysis of thecyclotetrasaccharide in a crystalline powder gave a thermogravimetriccurve in FIG. 49. Based on the relationship between the weight changeand the temperature, it was found that the weight reductioncorresponding to one mole of water was observed at a temperature ofabout 270° C. and further observed the weight reduction corresponding tothe decomposition of cyclotetrasaccharide per se at a temperature ofabout 290° C. or higher. These results confirmed that thecyclotetrasaccharide crystal in this experiment wascyclotetrasaccharide, monohydrate.

EXPERIMENT 32 Conversion into Anhydrous Crystal

Cyclotetrasaccharide, penta- or hexa-hydrate, in a crystalline powderform, obtained by the method in Experiment 30, was dried in vacuo at 40°C. or 120° C. for 16 hours. The Karl Fischer method of the resultingcrystalline powders revealed that the one dried at 40° C. had a moisturecontent of about 4.2%, while the other dried at 120° C. had a moisturecontent of about 0.2%, meaning that it was substantially anhydrous.Unlike quite different from the results from powder x-ray diffractionanalyses of the cyclotetrasaccharide, penta- or hexa-hydrate, and thecyclotetrasaccharide, monohydrate, before drying in vacuo, the powderx-ray analysis of the above cyclotetrasaccharide dried in vacuo at 40°and 120° C. gave characteristic diffraction spectra having maindiffraction angles (2θ) of 10.8°, 14.7°, 15.0°, 15.7°, and 21.5° in FIG.50 for 40° C. and FIG. 51 for 120° C. Although there found difference inpeak levels between the two diffraction spectra, they had substantiallythe same peak diffraction angles and they were crystallographicallyjudged to be substantially the same crystalline monohydrate. The factthat the base lines of the diffraction spectra exhibited a mountain-likepattern and the crystallinity of the crystalline monohydrate was lowerthan those of cyclotetrasaccharide, penta- or hexa-hydrate, andcyclotetrasaccharide, monohydrate, before drying in vacuo revealed thatthere existed an amorphous cyclotetrasaccharide. Based on this, thecyclotetrasaccharide powder with a moisture content of about 4.2%,obtained by drying in vacuo at 40° C., was estimated to be a mixturepowder of an amorphous cyclotetrasaccharide with such a moisture contentand anhydrous crystalline cyclotetrasaccharide. These data revealed thatcyclotetrasaccharide, penta- or hexa-hydrate, was converted intoamorphous and anhydrous forms when dried in vacuo. The thermogravimetricanalysis of anhydrous cyclotetrasaccharide with a moisture content of0.2%, which was conducted similarly as in Experiment 31, observed only aweight reduction as shown in FIG. 52, deemed to be induced by the heatdecomposition at a temperature of about 270° C. or higher as shown inFIG. 52.

EXPERIMENT 33 Saturation Concentration of Cyclotetrasaccharide in Water

To study the saturation concentration of cyclotetrasaccharide in waterat 10-90° C., 10 ml of water was placed in a glass vessel with a sealcap, and then mixed with cyclotetrasaccharide, penta- or hexa-hydrate,obtained by the method in Experiment 30, in an excessive amount over alevel dissolving completely at respective temperatures, cap-sealed, andstirred for two days while keeping at respective temperatures of 10-90°C. until being saturated. Each resulting saturated solution ofcyclotetrasaccharide was membrane filtered to remove undissolvedcyclotetrasaccharide, and each filtrate was then examined for moisturecontent by the drying loss method to determine a saturationconcentration of cyclotetrasaccharide at respective temperatures. Theresults are in Table 34. TABLE 34 Temperature (° C.) Saturationconcentration (%) 10 30.3 30 34.2 50 42.6 70 53.0 90 70.5

EXPERIMENT 34 Thermostability

A crystalline cyclotetrasaccharide, penta- or hexa-hydrate, obtained bythe method in Experiment 30, was dissolved in water into a 10% (w/v)aqueous solution of cyclotetrasaccharide, and eight milliliters of whichwas placed in a glass test tube, followed by sealing the test tube andheating the aqueous solution at 120° C. for 30-90 min. After theheating, the aqueous solution was cooled under atmospheric conditionsand measured for coloration degree and determined for purity on HPLC.The coloration degree was evaluated based on the absorbance in a cellwith a 1-cm light pass at a wavelength of 480 nm. The results are inTable 35. TABLE 35 Heating time Coloration degree Purity (min)(A_(480 nm)) (%) 0 0.00 100 30 0.00 100 60 0.00 100 90 0.00 100

As evident from the results in Table 35, it was revealed thatcyclotetrasaccharide is a thermostable saccharide because an aqueoussolution of cyclotetrasaccharide was not colored and the purity of thesaccharide composition was not lowered even when heated at a hightemperature of 120° C.

EXPERIMENT 35 pH Stability

A crystalline cyclotetrasaccharide, penta- or hexa-hydrate, obtained bythe method in Experiment 30, was dissolved in 20 mM buffers withdifferent pHs into a 4% (w/v) cyclotetrasaccharide solution with a pH of2-10. Eight milliliters of each solution was placed in a glass testtube, followed by sealing the test tube and heating the solution at 100°C. for 24 hours. After cooling, each solution was measured forcoloration degree and determined for purity on HPLC. The colorationdegree was evaluated based on the absorbance in a cell with a 1-cm lightpass at a wavelength of 480 nm. The results are in Table 36. TABLE 36 pHColoration degree Purity (type of buffer) (A_(480 nm)) (%)  2.0 (Acetatebuffer) 0.00 93  3.0 (Acetate buffer) 0.00 100  4.0 (Acetate buffer)0.00 100  5.0 (Acetate buffer) 0.00 100  6.0 (Tris-HCl buffer) 0.00 100 7.0 (Tris-HCl buffer) 0.00 100  8.0 (Tris-HCl buffer) 0.00 100  9.0(Ammonium buffer) 0.00 100 10.0 (Ammonium buffer) 0.00 100

As evident from the results in Table 36, an aqueous solution ofcyclotetrasaccharide was not colored even when heated at 100° C. for 24hours in a wide pH range from 2 to 10, and the purity of the saccharidecomposition was not lowered at all in a pH range from 3 to 10, eventhough the purity was slightly lowered at pH 2, and these facts revealedthat cyclotetrasaccharide was highly stable in a relatively wide pHrange, i.e., an acid pH range from 3 to 5, a neutral pH range from 6 to8, and an alkaline pH range from 9 to 10.

EXPERIMENT 36 Amino Carbonyl Reaction

A crystalline cyclotetrasaccharide, penta- or hexa-hydrate, obtained bythe method in Experiment 30, was dissolved in water, and then admixedwith commercialized special grade glycine and phosphate buffer, and theresulting mixture was then adjusted to pH 7.0 with 50 mM phosphatebuffer to obtain a 10% (w/v) cyclotetrasaccharide solution containing 1%(w/v) glycine. Four milliliters of the resulting solution were placed ina glass test tube, sealed, and heated at 100° C. for 30 to 90 min. Afterallowing to stand for cooling at ambient temperature, each of theresulting solutions was measured for coloration degree to examine ontheir amino carbonyl reactivity. The coloration degree was evaluatedbased on the absorbance in a cell with 1-cm light pass at a wavelengthof 480 nm. The results are in Table 37. TABLE 37 Heating time (min)Coloration degree (A_(480 nm)) 0 0.00 30 0.00 60 0.00 90 0.00

As evident from the results in Table 37, cyclotetrasaccharide was notcolored even when heated in the presence of glycine, meaning that thesaccharide does not induce browning with glycine, i.e.,cyclotetrasaccharide is a stable saccharide which does not induce theamino carbonyl reaction, alias the Maillard reaction.

EXPERIMENT 37 Amino Carbonyl Reaction

A crystalline cyclotetrasaccharide, penta- or hexa-hydrate, obtained bythe method in Experiment 30, and a commercialized polypeptone,Nihonseiyaku K.K., Tokyo, Japan, were dissolved in deionized water toobtain a 10% (w/v) cyclotetrasaccharide solution containing 5% (w/v)polypeptone. Four milliliters of the resulting solution were placed in aglass test tube, sealed, and heated at 100° C. for 30 to 90 min. Afterallowing to stand for cooling at ambient temperature, each of theresulting solutions was measured for coloration degree to examine ontheir amino carbonyl reactivity. In parallel, as a control, a solutionwith only polypeptone was provided and similarly treated as above. Thecoloration degree was evaluated based on the level of the absorbance,measured in a cell with 1-cm light pass at a wavelength of 480 nm, minusthe control. The results are in Table 38. TABLE 38 Heating time (min)Coloration degree (A_(480 nm)) 0 0.00 30 0.00 60 0.00 90 0.00

As evident from the results in Table 38, it was revealed thatcyclotetrasaccharide did not induce browning with polypeptone whenheated in the presence of polypeptone, i.e., the saccharide is a stablesaccharide which substantially does not induce the amino carbonylreaction.

EXPERIMENT 38 Inclusion Action

A crystalline cyclotetrasaccharide, penta- or hexa-hydrate, obtained bythe method in Experiment 30, was dissolved in deionized water to obtaina 20% (w/v) aqueous solution of cyclotetrasaccharide. To 100 g of theaqueous solution was added 2 g of methanol, 3 g of ethanol, or 4.6 gacetic acid to be included by the cyclotetrasaccharide. Thereafter, eachof the resulting solutions was filtered to remove non-included products,and the filtrate was dried in vacuo. As a control, similar inclusionproducts were prepared by using “ISOELITE™ P”, a branched cyclodextrincommercialized by Maruha K.K., Tokyo, Japan, which were known to haveinclusion ability.

To measure the amount of the inclusion products in the resultinglyophilized powders, one gram of each powder was dissolved in fivemilliliters water and extracted after admixing with five milliliters ofdiethylether. The extraction was repeated, and the resulting extractswere quantified on gas chromatography. The results are in Table 39.TABLE 39 Inclusion Inclusion amount (mg/g lyophilized powder) productCyclotetrasaccharide ISOELITE P (control) Methanol 6.71 2.92 Ethanol17.26 8.92 Acetic acid 67.74 30.57

As evident from the results in Table 39, it was revealed thatcyclotetrasaccharide has inclusion ability about 2-folds higher thanthat of the branched cyclodextrin by weight.

EXPERIMENT 39 Sweetening Power

A crystalline cyclotetrasaccharide, penta- or hexa-hydrate, obtained bythe method in Experiment 30, was dissolved in deionized water to obtain10-30% (w/v) aqueous solutions of cyclotetrasaccharide for testsolutions on sweetening power. Using a 6% (w/v) aqueous solution of acommercialized granulated sugar as a standard, a sensory test with eightpanelists was conducted. As a result, the sweetening power ofcyclotetrasaccharide was about 27% of that of sucrose.

EXPERIMENT 40 Digestion Test

Using a crystalline cyclotetrasaccharide, penta- or hexa-hydrate,obtained by the method in Experiment 30, the digestibility ofcyclotetrasaccharide in vitro by salivary amylase, synthetic gastricjuice, amylopsin, and intestinal mucosal enzyme was carried out inaccordance with the method as reported by K. Okada et al. in JOURNAL OFJAPANESE SOCIETY OF NUTRITION AND FOOD SCIENCE, Vol. 43, No. 1, pp.23-29 (1990). As a control, maltitol known as a substantiallynon-digestive saccharide was used. The results are in Table 40. TABLE 40Decomposition percentage (%) by digestive enzyme Maltitol Digestiveenzyme Cyclotetrasaccharide (Control) Salivary amylase 0.0 0.0 Synthetic0.0 0.0 gastric juice Amylopsin 0.0 0.0 Small intestinal 0.74 4.0mucosal enzyme

As evident from the results in Table 40, cyclotetrasaccharide was notcompletely digested by salivary amylase, synthetic gastric juice, andamylopsin, but slightly digested by intestinal mucosal enzyme at adigestibility as low as 0.74% corresponding to ⅕ of that of maltitol asa control. These results confirmed that cyclotetrasaccharide is a highlyundigestible saccharide.

EXPERIMENT 41 Fermentation Test

Using a crystalline cyclotetrasaccharide, penta- or hexa-hydrate,obtained by the method in Experiment 30, the fermentability ofcyclotetrasaccharide by an internal content of rat cecum was tested inaccordance with the method by T. Oku in “Journal of Nutritional Scienceand Vitaminology”, Vol. 37, pp. 529-544 (1991). The internal content ofrat cecum was collected by anesthetizing a Wister male rat with ether,allowing the rat to die, collecting the internal content under anaerobicconditions, and suspending the resultant with 4-fold volumes of a 0.1 Maqueous solution of sodium bicarbonate. Cyclotetrasaccharide was addedin an amount of about 7% by weight to the internal content of rat cecum,and the contents of cyclotetrasaccharide still remained just after and12 hours after the addition of the internal content was quantified ongas chromatography. As a result, the contents of cyclotetrasaccharide ofthe former and latter were respectively 68.0 mg and 63.0 mg per one gramof the internal content of rat cecum. These data confirmed thatcyclotetrasaccharide is a substantially non-fermentable saccharide.

EXPERIMENT 42 Assimilation Test

Using a crystalline cyclotetrasaccharide, penta- or hexa-hydrate,obtained by the method in Experiment 30, the assimilability ofcyclotetrasaccharide by an internal content of rat cecum was studied inaccordance with the method disclosed in “A Color Atlas of AnaerobicBacteria”, edited by Tomotari MITSUOKA, published by Kabushiki KaishaSobunsha, Tokyo, Japan, (1984). About 10⁷ CFU (colony forming units) ofpre-cultured fresh microorganisms were inoculated into five millilitersof PYF medium admixed with 0.5% cyclotetrasaccharide, and cultured at37° C. for four days under anaerobic conditions. As a control, glucosewas used as an easily assimilable saccharide. The assimilability wasjudged negative (−) when the post culture had a pH of 6.0 or higher andjudged positive (+) when the post culture had a pH below 6.0. Thejudgement of assimilability was confirmed by measuring the content ofsaccharide, remained in the culture, using the anthrone method todetermine the lowered saccharide level. The results are in Table 41.TABLE 41 Strain of intestinal Assimilability MicroorganismCyclotetrasaccharide Glucose (control) Bacteroides vulgatus − + JCM 5826Bifidobacterium adolescentis − + JCM 1275 Clostridium perfringens − +JCM 3816 Escherichia coli − + IFO 3301 Eubacterium aerofaciens − + ATCC25986 Lactobacillus acidophilus − + JCM 1132

As evident from the results in Table 41, it was confirmed thatcyclotetrasaccharide was not assimilated by all the strains tested, butglucose as a control was assimilated by all the strains tested. Thus,cyclotetrasaccharide was confirmed to be a highly non-assimilablesaccharide by intestinal microorganisms.

EXPERIMENT 43 Acute Toxicity Test

The acute toxicity of a crystalline cyclotetrasaccharide, penta- orhexa-hydrate, obtained by the method in Experiment 30, was tested byorally administering it to mice. As a result, it was revealed thatcyclotetrasaccharide had relatively low toxicity and did not inducedeath of mice even when administered at a highest possible dose. Basedon this, the LD₅₀ of cyclotetrasaccharide was at least 50 g/kg mousebody weight, though the data were not so accurate.

Based on the results in Experiments 40 to 43, cyclotetrasaccharide isnot substantially assimilated or absorbed by living bodies when orallytaken and can be expected to be used as a non- or low-caloric ediblematerial in diet sweeteners, fillers for sweeteners with a relativelyhigh sweetening power, and viscosity agents, fillers and bodies for dietfood products, and further can be used as an edible fiber and foodmaterial for substituting fats.

The following Example A describes the cyclotetrasaccharide and theprocess for producing saccharide composition comprising the same, andExample B describes the composition comprising the cyclotetrasaccharideor the saccharide composition:

EXAMPLE A-1

A microorganism of the species Bacillus globisporus C9, FERM BP-7143,was cultured by a fermentor for 48 hours in accordance with the methodin Experiment 3. After completion of the culture, the resulting culturewas filtered with an SF membrane to remove cells and to collect about 18L of a culture supernatant. Then the culture supernatant wasconcentrated with a UF membrane to collect about one liter of aconcentrated enzyme solution containing 8.8 units/ml of theα-isomaltosylglucosaccharide-forming enzyme of the present invention and26.7 units/ml of α-isomaltosyl-transferring enzyme.

A potato starch was prepared into an about 2% starch suspension, admixedwith calcium chloride to give a final concentration of 1 mM, adjusted topH 6.0, and heated at 95° C. for about 20 min to gelatinize the starch.The resulting mixture was then cooled to about 35° C. and admixed with0.25 ml of the above concentrated enzyme solution to one gram of thestarch, d.s.b., followed by the enzymatic reaction at pH 6.0 and 35° C.for 48 hours. The reaction mixture was heated to and kept at 95° C. for10 min, and then cooled and filtered. The filtrate was in a conventionalmanner decolored with an activated charcoal, desalted and purified withion exchangers in H- and OH-forms, and further concentrated andspray-dried to obtain a powder containing cyclotetrasaccharide in ayield of about 90% to the material starch, d.s.b.

Since the product contains, on a dry solid basis, 0.7% glucose, 1.4%isomaltose, 11.1% maltose, 62.1% cyclotetrasaccharide, and 24.7% ofother saccharides and has a mild sweetness and an adequate viscosity,moisture-retaining ability, and inclusion ability, it can beadvantageously used in a variety of compositions such as food products,cosmetics, and pharmaceuticals as a sweetener, taste-improving agent,quality-improving agent, syneresis-preventing agent, stabilizer,discoloration-preventing agent, filler, inclusion agent, and base forpulverization.

EXAMPLE A-2

A potato starch was prepared into an about 6% starch suspension, admixedwith calcium carbonate to give a final concentration of 0.1%, adjustedto pH 6.0, further admixed with 0.2% per gram starch, d.s.b., of“TERMAMYL 60L”, an α-amylase commercialized by Novo Industri A/S,Copenhagen, Denmark, and then heated at 95° C. for about 10 min.Thereafter, the mixture was autoclaved at 120° C. for 20 min and thenpromptly cooled to about 35° C. to obtain a liquefied solution with a DE(dextrose equivalent) of about four. To the liquefied solution was added0.25 ml per gram starch, d.s.b., of the concentrated enzyme solution inExample A-1 containing α-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme, followed by the enzymatic reaction atpH 6.0 and 35° C. for 48 hours. The reaction mixture was heated to andkept at 95° C. for 10 min and then cooled and filtered. The filtrate wasin a conventional manner decolored with an activated charcoal, desaltedand purified with ion exchangers in H- and OH-forms, and thenconcentrated into a 60% cyclotetrasaccharide syrup in a yield of about90% to the material starch, d.s.b.

Since the product contains, on a dry solid basis, 0.9% glucose, 1.5%isomaltose, 11.3% maltose, 60.1% cyclotetrasaccharide, and 26.2% ofother saccharides and has a mild sweetness and an adequate viscosity,moisture-retaining ability, and inclusion ability, it can beadvantageously used in a variety of compositions such as food products,cosmetics, and pharmaceuticals as a sweetener, taste-improving agent,quality-improving agent, syneresis-preventing agent, stabilizer,discoloration-preventing agent, filler, and inclusion agent.

EXAMPLE A-3

A microorganism of the species Bacillus globisporus C11, FERM BP-7144,was cultured by a fermentor for 48 hours in accordance with the methodin Experiment 6. After completion of the culture, the resulting culturewas filtered with an SF membrane to remove cells and to collect about 18L of a culture supernatant. Then the culture supernatant wasconcentrated with a UF membrane to collect about one liter of aconcentrated enzyme solution containing 9.0 units/ml of theα-isomaltosylglucosaccharide-forming enzyme of the present invention and30.2 units/ml of α-isomaltosyl-transferring enzyme. A tapioca starch wasprepared into an about 25% starch suspension which was then admixed with0.2% per gram starch, d.s.b., of “NEO-SPITASE”, an α-amylasecommercialized by Nagase Biochemicals, Ltd., Kyoto, Japan. Thereafter,the reaction mixture was autoclaved at 120° C. for 20 min and thenpromptly cooled to about 35° C. to obtain a liquefied solution with a DEof about four. To the liquefied solution was added 0.25 ml per gramstarch, d.s.b., of the above concentrated enzyme solution, containingα-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme, and further added 10 units/g starch,d.s.b., of CGTase commercialized by Hayashibara BiochemicalLaboratories, Inc., Okayama, Japan, followed by the enzymatic reactionat pH 6.0 and 35° C. for 48 hours. The reaction mixture was heated toand kept at 95° C. for 30 min and then cooled and filtered, and thenadjusted to pH 5.0 and 50° C. and admixed with 300 units/g starch,d.s.b., of “TRANSGLUCOSIDASE L AMANO™”, an α-glucosidase commercializedby Amano Pharmaceutical Co., Ltd., Aichi, Japan, followed by theenzymatic reaction for 24 hours. Further the reaction mixture was mixedwith 30 units/g starch, d.s.b., “GLUCOZYME”, a glucoamylase preparationcommercialized by Nagase Biochemicals, Ltd., Kyoto, Japan, and thenenzymatically reacted for 17 hours. The reaction mixture thus obtainedwas heated to and kept at 95° C. for 30 min, and then cooled andfiltered to obtain a filtrate. The resulting filtrate was in aconventional manner decolored with an activated charcoal, desalted andpurified with ion exchangers in H- and OH-forms, and then concentratedinto a 60% cyclotetrasaccharide syrup in a yield of about 90% to thematerial starch, d.s.b.

Since the product contains, on a dry solid basis, 38.4% glucose, 58.1%cyclotetrasaccharide, and 3.5% of other saccharides and has a mildsweetness and an adequate viscosity, moisture-retaining ability, andinclusion ability, it can be advantageously used in a variety ofcompositions such as food products, cosmetics, and pharmaceuticals as asweetener, taste-improving agent, quality-improving agent,syneresis-preventing agent, stabilizer, discoloration-preventing agent,filler, and inclusion agent.

EXAMPLE A-4

A potato starch was prepared into an about 20% starch suspension,admixed with calcium carbonate to give a final concentration of 0.1%,adjusted to pH 6.5, further admixed with 0.3% per gram starch, d.s.b.,of “TERMAMYL 60L”, an α-amylase commercialized by Novo Industri A/S,Copenhagen, Denmark, and then enzymatically reacted at 95° C. for about15 min. Thereafter, the mixture was autoclaved at 120° C. for 20 min andthen promptly cooled to about 35° C. to obtain a liquefied solution witha DE of about four. To the liquefied solution was added 0.25 ml per gramstarch, d.s.b., of the concentrated enzyme solution in Example A-3containing α-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme, followed by the enzymatic reaction atpH 6.0 and 35° C. for 48 hours. The reaction mixture was heated to andkept at 95° C. for 30 min and then adjusted to pH 5.0 and 50° C.,followed by the enzymatic reaction for 24 hours after the addition of300 units/g solid of “TRANSGLUCOSIDASE L AMANO™”, an α-glucosidasecommercialized by Amano Pharmaceutical Co., Ltd., Aichi, Japan, and thenthe enzymatic reaction for 17 hours after the addition of 30 units/gsolid of “GLUCOZYME”, a glucoamylase preparation commercialized byNagase Biochemicals, Ltd., Kyoto, Japan. The resulting reaction mixturewas heated to and kept at 95° C. for 30 min, and then cooled andfiltered. The filtrate thus obtained was in a conventional mannerdecolored with an activated charcoal, desalted and purified with ionexchangers in H- and OH-forms, and then concentrated into a 60%cyclotetrasaccharide syrup in a yield of about 90% to the materialstarch, d.s.b.

Since the product contains, on a dry solid basis, 34.2% glucose, 62.7%cyclotetrasaccharide, and 3.1% of other saccharides and has a mildsweetness and an adequate viscosity, moisture-retaining ability, andinclusion ability, it can be advantageously used in a variety ofcompositions such as food products, cosmetics, and pharmaceuticals as asweetener, taste-improving agent, quality-improving agent,syneresis-preventing agent, stabilizer, discoloration-preventing agent,filler, and inclusion agent.

EXAMPLE A-5

Cyclotetrasaccharide syrup obtained by the method in Example A-3 wascolumn chromatographed using “AMBERLITE CR-1310 (Na-form)”, a strongacid cation-exchange resin commercialized by Japan Organo Co., Ltd.,Tokyo, Japan. The resin was packed into four jacketed stainless steelcolumns having a diameter of 5.4 cm, which were then cascaded in seriesto give a total gel bed depth of 20 m. Under the conditions of keepingthe inner column temperature at 60° C., the saccharide syrup was fed tothe columns in a volume of 5% (v/v) and fractionated by feeding to thecolumns hot water heated to 60° C. at an SV (space velocity) of 0.13 toobtain high cyclotetrasaccharide content fractions while monitoring thesaccharide composition of eluate on HPLC, and then purifying thefractions to obtain a high cyclotetrasaccharide content solution in ayield of about 21% to the material starch, d.s.b. The solution containedabout 98%, d.s.b., of cyclotetrasaccharide.

The solution was concentrated to give a concentration of about 70% andthen placed in a crystallizer, admixed with about 2% of crystallinecyclotetrasaccharide, penta- or hexa-hydrate, and gradually cooled toobtain a massecuite with a crystallinity of about 45%. The massecuitewas sprayed from a nozzle equipped on top of a drying tower at a highpressure of 150 kg/cm². Simultaneously, hot air heated to 85° C. wasbeing blown down from the upper part of the drying tower, and theresulting crystalline powder was collected on a transporting wireconveyor provided on the basement of the tower and gradually moved outof the tower while blowing thereunto a hot air heated to 45° C. Theresulting crystalline powder was injected to an ageing tower and agedfor 10 hours while a hot air was being blown to the contents to completethe crystallization and drying to obtain a crystalline powder ofcyclotetrasaccharide, penta- or hexa-hydrate.

Since the product has a relatively low reducibility, does substantiallyneither cause the amino carbonyl reaction nor exhibit hygroscopicity,and has a satisfactory handleability, mild low sweetness, adequateviscosity, moisture-retaining ability, inclusion ability, andsubstantially non-digestibility, it can be advantageously used in avariety of compositions such as food products, cosmetics, andpharmaceuticals as a sweetener, materials for relatively low caloricfoods, taste-improving agent, flavor and taste-improving agent,quality-improving agent, syneresis-preventing agent, stabilizer,discoloration-preventing agent, filler, inclusion agent, and base forpulverization.

EXAMPLE A-6

To increase the content of cyclotetrasaccharide of cyclotetrasaccharidesyrup obtained by the method in Example A-4, the syrup as a materialsaccharide solution was column chromatographed using a strong acidcation-exchange resin in accordance with the method in Example A-5,followed by collecting and purifying high cyclotetrasaccharide contentfractions to obtain a high cyclotetrasaccharide content solution in ayield of about 90% to the material starch, d.s.b.

The solution was concentrated to give a concentration of about 85% andthen gradually cooled while stirring to proceed crystallization. Theresultant was transferred to a plastic vessel and allowed to stand atambient temperature for crystallizing and ageing the contents. Theresulting block was pulverized by a cutter to obtain a crystallinepowder of cyclotetrasaccharide, penta- or hexa-hydrate.

Since the product does not substantially have hygroscopicity, has asatisfactory handleability, mild low sweetness, adequate viscosity,moisture-retaining ability, inclusion ability, and substantiallynon-digestibility, it can be advantageously used in a variety ofcompositions such as food products, cosmetics, and pharmaceuticals as asweetener, materials for relatively low caloric foods, taste-improvingagent, flavor and taste-improving agent, quality-improving agent,syneresis-preventing agent, stabilizer, discoloration-preventing agent,filler, inclusion agent, and base for pulverization.

EXAMPLE A-7

A high cyclotetrasaccharide content solution, obtained by the method inExample A-6, was continuously crystallized while concentrating. Theresulting massecuite was separated by a basket-type centrifuge to obtaincrystals which were then sprayed with a small amount of water to obtaina high purity cyclotetrasaccharide, penta- or hexa-hydrate, in a yieldof about 55%, d.s.b., to the material contents.

Since the product contains at least 98%, d.s.b., of a high puritycrystalline cyclotetrasaccharide, penta- or hexa-hydrate, has arelatively low reducibility, does substantially neither cause the aminocarbonyl reaction nor exhibit hygroscopicity, and has a satisfactoryhandleability, mild low sweetness, adequate viscosity,moisture-retaining ability, inclusion ability, and substantiallynon-digestibility, it can be advantageously used in a variety ofcompositions such as food products, cosmetics, pharmaceuticals,industrial reagents, and chemical materials as a sweetener, materialsfor relatively low caloric foods, taste-improving agent, flavor andtaste-improving agent, quality-improving agent, syneresis-preventingagent, stabilizer, discoloration-preventing agent, filler, inclusionagent, and base for pulverization.

EXAMPLE A-8

A liquid nutrient culture medium, consisting of 5.0% (w/v) cornphytoglycogen, 1.0% (w/v) of “ASAHIMEAST”, a yeast extract, 0.1% (w/v)of dipotassium phosphate, 0.06% (w/v) of sodium phosphate dodecahydrate,0.05% (w/v) magnesium sulfate heptahydrate, and water, was placed in a30-L fermentor in a volume of about 20 L, autoclaved at 121° C. for 20minutes to effect sterilization, cooled to 27° C., inoculated with 1%(v/v) of a seed culture of Bacillus globisporus C11, FERM BP-7144,prepared in accordance with the method in Experiment 6, and incubated at27° C. and pH 6.0-7.0 for 72 hours under aeration and agitationconditions. The resultant culture was sterilized by heating at 121° C.for 20 min, cooled, and centrifuged. The supernatant was collected andmembrane filtered with a UF membrane. The resulting filtrate was in ausual manner decolored with an activated charcoal and desalted andpurified with ion exchangers in H- and OH-forms to obtain a solutioncontaining cyclotetrasaccharide in a yield of about 40%, d.s.b., to thematerial phytoglycogen. The solution thus obtained contained about 87%,d.s.b., of cyclotetrasaccharide.

The above solution was continuously crystallized while concentrating,and the resulting massecuite was separated by a basket-type centrifugeto obtain crystals which were then sprayed with a small amount of waterto obtain cyclotetrasaccharide, penta- or hexa-hydrate, with a purity ofat least 98% in a yield of about 25%, d.s.b., to the materialphytoglycogen.

Since the product, a high purity cyclotetrasaccharide, penta- orhexa-hydrate, has a relatively low reducibility, does substantiallyneither cause the amino carbonyl reaction nor exhibit hygroscopicity,and has a satisfactory handleability, mild low sweetness, adequateviscosity, moisture-retaining ability, inclusion ability, andsubstantially non-digestibility, it can be advantageously used in avariety of compositions such as food products, cosmetics,pharmaceuticals, industrial reagents, and chemical materials as asweetener, materials for relatively low caloric foods, taste-improvingagent, flavor and taste-improving agent, quality-improving agent,syneresis-preventing agent, stabilizer, discoloration-preventing agent,filler, inclusion agent, and base for pulverization.

EXAMPLE A-9

A microorganism of the species Bacillus globisporus N75, FERM BP-7591,was cultured by a fermentor for 48 hours in accordance with the methodin Experiment 10. After completion of the culture, the resulting culturewas filtered with an SF membrane to remove cells and to collect about 18L of a culture supernatant. Then the culture supernatant wasconcentrated with a UF membrane to collect about 800 ml of aconcentrated enzyme solution containing 6.0 units/ml of theα-isomaltosylglucosaccharide-forming enzyme of the present invention and20.0 units/ml of α-isomaltosyl-transferring enzyme. A corn starch wasprepared into an about 30% starch suspension which was then admixed withcalcium carbonate to give a concentration of 0.1%, adjusted to pH 6.5,admixed with 0.3% per gram starch, d.s.b., of “TERMAMYL 60L”, anα-amylase commercialized by Novo Industri A/S, Copenhagen, Denmark,”, anα-amylase commercialized by Nagase Biochemicals, Ltd., Kyoto, Japan, andenzymatically reacted at 95° C. for 15 min. Thereafter, the reactionmixture was autoclaved at 120° C. for 20 min and then promptly cooled toabout 51° C. to obtain a liquefied solution with a DE of four. To theliquefied solution were added 0.4 ml per gram of the starch, d.s.b., ofthe above concentrated enzyme solution containingα-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme, and three units/g starch, d.s.b., ofCGTase commercialized by Hayashibara Biochemical Laboratories, Inc.,Okayama, Japan, followed by the enzymatic reaction at pH 5.5 and 51° C.for 48 hours. Thereafter, the reaction mixture was heated to and kept at95° C. for 30 min, then adjusted to pH 5.0 and 50° C., followed by the24-hour enzymatic reaction after the addition of 300 units/g solid of“TRANSGLUCOSIDASE L AMANO™”, an α-glucosidase commercialized by AmanoPharmaceutical Co., Ltd., Aichi, Japan, and then the 17-hour enzymaticreaction after the addition of 20 units/g solid of “GLUCOZYME”, aglucoamylase preparation commercialized by Nagase Biochemicals, Ltd.,Kyoto, Japan. The resulting reaction mixture was heated to and kept at95° C. for 30 min, cooled, and filtered. The resulting filtrate was in ausual manner decolored with an activated charcoal, desalted and purifiedwith ion-exchangers in H- and OH-forms, and concentrated to obtain asyrup containing 44.0%, d.s.b., of cyclotetrasaccharide. To increase thecontent of cyclotetrasaccharide, the syrup as a material saccharidesolution was column chromatographed using a strong acid cation-exchangeresin in accordance with the method in Example A-5, followed bycollecting and purifying high cyclotetrasaccharide content fractions andthen concentrating and spray drying the resultant to obtain a powdercontaining cyclotetrasaccharide in a yield of about 45%, d.s.b., to thematerial starch.

Since the product contains 3.7% glucose, 80.5% cyclotetrasaccharide, and15.8% other saccharides, has a mild low sweetness, adequate viscosity,moisture-retaining ability, and inclusion ability, it can beadvantageously used in a variety of compositions such as food products,cosmetics, pharmaceuticals as a sweetener, taste-improving agent,quality-improving agent, syneresis-preventing agent, stabilizer,discoloration-preventing agent, filler, inclusion agent, and base forpulverization.

EXAMPLE A-10

A microorganism of the species Bacillus globiformis A19, FERM BP-7590,was cultured by a fermentor for 48 hours in accordance with the methodin Experiment 14. After completion of the culture, the resulting culturewas filtered with an SF membrane to remove cells and to collect about 18L of a culture supernatant. Then the culture supernatant wasconcentrated with a UF membrane to collect about one liter of aconcentrated enzyme solution containing 15.2 units/ml of theα-isomaltosylglucosaccharide-forming enzyme of the present invention and23.0 units/ml of α-isomaltosyl-transferring enzyme.

A potato starch was prepared into an about 5% starch suspension whichwas then admixed with calcium carbonate to give a concentration of 0.1%,adjusted to pH 6.0, admixed with 0.2% per gram starch, d.s.b., of“TERMAMYL 60L”, an α-amylase commercialized by Novo Industri A/S,Copenhagen, Denmark, and enzymatically reacted at 95° C. for 10 min.Thereafter, the reaction mixture was autoclaved at 120° C. for 20 minand then promptly cooled to about 40° C. to obtain a liquefied solutionwith a DE of four. To the liquefied solution were added 0.5 ml per gramof the starch, d.s.b., of the concentrated enzyme solution containingα-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme obtained by the above method, and thenenzymatically reacted at pH 6.0 and 40° C. for 48 hours. The reactionmixture was heated to and kept at 95° C. for 10 min, then cooled andfiltered. The filtrate was in a usual manner decolored with an activatedcharcoal, desalted and purified with ion-exchangers in H- and OH-forms,and concentrated to obtain a syrup containing 70% (w/v)cyclotetrasaccharide in a yield of about 90%, d.s.b., to the materialstarch.

Since the product contains 2.5% glucose, 6.3% isomaltose, and 30.1%cyclotetrasaccharide, has a mild sweetness, adequate viscosity,moisture-retaining ability, and inclusion ability, it can beadvantageously used in a variety of compositions such as food products,cosmetics, pharmaceuticals as a sweetener, taste-improving agent,quality-improving agent, syneresis-preventing agent, stabilizer,discoloration-preventing agent, filler, and inclusion agent.

EXAMPLE B-1 Sweetener

To 0.8 part by weight of a crystalline tetrasaccharide, penta- orhexa-hydrate, obtained by the method in Example A-7, were homogeneouslyadded 0.2 part by weight of “TREHA^(□)”, a crystalline trehalose hydratecommercialized by Hayashibara Shoji Inc., Okayama, Japan, 0.01 part byweight of “αG SWEET™”, (α-glycosylstevioside commercialized by ToyoSugar Refining Co., Tokyo, Japan), and 0.01 part by weight of“ASPARTAME” (L-aspartyl-L-phenylalanine methyl ester), and the resultingmixture was fed to a granulator to obtain a sweetener in a granule form.The product has a satisfactory sweetness and a 2-fold higher sweeteningpower of sucrose. Since crystalline cyclotetrasaccharide, penta- orhexa-hydrate, is scarcely digested and fermented and is substantiallyfree of calorie, the calorie of the product is about 1/10 of that ofsucrose with respect to sweetening power. In addition, the product issubstantially free from deterioration and stable when stored at ambienttemperature. Thus, the product can be suitably used as a high qualitylow-caloric and less cariogenic sweetener.

EXAMPLE B-2 Hard Candy

One hundred parts by weight of a 55% (w/v) sucrose solution was admixedwhile heating with 50 parts by weight of a syrup containingcyclotetrasaccharide obtained by the method in Example A-2. The mixturewas then concentrated by heating under reduced pressure to give amoisture content of less than 2%, and the concentrate was mixed with 0.6part by weight of citric acid and an adequate amount of a lemon flavor,followed by forming in a usual manner the resultant into the desiredproduct. The product is a stable, high quality hard candy which has asatisfactory mouth feel, taste, and flavor, less adsorb moisture, anddoes neither induce crystallization of sucrose nor cause melting.

EXAMPLE B-3 Chewing Gum

Three parts by weight of a gum base were melted by heating to an extentto be softened and then admixed with two parts by weight of anhydrouscrystalline maltitol, two parts by weight of xylitol, two parts byweight of a crystalline cyclotetrasaccharide, penta- or hexa-hydrate,obtained by the method in Example A-7, and one part by weight oftrehalose, and further mixed with adequate amounts of a flavor and acolor. The mixture was in a usual manner kneaded by a roll and thenshaped and packed to obtain the desired product. The product thusobtained is a relatively low cariogenic and caloric chewing gum having asatisfactory texture, taste, and flavor.

EXAMPLE B-4 Sweetened Milk

In 100 parts by weight of a fresh milk was dissolved two parts by weightof a crystalline powder of cyclotetrasaccharide, penta- or hexa-hydrate,obtained by the method in Example A-5, and two parts by weight ofsucrose, and the solution was sterilized by heating using a plate heaterand then concentrated to give a concentration of 70%. The concentratewas aseptically canned to obtain the desired product. Since the producthas a mild sweetness and a satisfactory flavor and taste, it can bearbitrarily used for seasoning fruit, coffee, cocoa, and tea.

EXAMPLE B-5 Lactic Acid Beverage

One hundred and seventy-five parts by weight of a skim milk powder, 130parts by weight of a syrup containing cyclotetrasaccharide, obtained bythe method in Example A-4, and 50 parts by weight of “NYUKAOLIGO^(□)”, ahigh lactosucrose content powder commercialized by Hayashibara ShojiInc., Okayama, Japan, were dissolved in 1,150 parts by weight of water.The resulting solution was sterilized at 65° C. for 30 min, then cooledto 40° C., inoculated in a usual manner with 30 parts by weight oflactic acid bacteria as a starter, and incubated at 37° C. for eighthours to obtain a beverage with lactic acid bacteria. The product can besuitably used as a lactic acid beverage which has a satisfactory flavorand taste, contains oligosaccharides and cyclotetrasaccharide, stablyretains the lactic acid bacteria, and has actions of promoting thegrowth of the bacteria and control the intestinal conditions.

EXAMPLE B-6 Powdered Juice

Thirty-three parts by weight of an orange juice powder, prepared byspray drying, were well mixed by stirring with 50 parts by weight ofcyclotetrasaccharide, penta- or hexa-hydrate, obtained by the method inExample A-7, 10 parts by weight of anhydrous crystalline maltitol, 0.65part by weight of anhydrous citric acid, 0.1 part by weight of malicacid, 0.2 part by weight of 2-O-α-D-glucosyl-L-ascorbic acid, 0.1 partby weight of sodium citrate, 0.5 part by weight of pullulan, and anadequate amount of a powdered flavor. The mixture was pulverized into aminute powder which was then placed in a fluidized-bed granulatoradjusted to blow air to 40° C., sprayed with an adequate amount of aconcentrated solution enriched with cyclotetrasaccharide as a binder,obtained by the method in Example A-5, granulated for 30 min, weighed,and packed to obtain the desired product. The product is a powderedjuice having a fruit juice content of about 30%. Also the product has ahigh product value as a high quality, low caloric juice because it hasno unpleasant taste and smell.

EXAMPLE B-7 Custard Cream

One hundred parts by weight of corn starch, 100 parts by weight ofcyclotetrasaccharide obtained by the method in Example A-2, 60 parts byweight of trehalose, 40 parts by weight of sucrose, and one part byweight of salt were sufficiently mixed, and then further mixed with 280parts by weight of fresh eggs, followed by stirring. To the resultingmixture was gradually admixed with 1,000 parts by weight of a boilingmilk. The mixture was continued stirring over a fire, and the heatingwas stopped when the whole contents became semitransparent after thecorn starch was completely gelatinized, followed by cooling theresultant, admixing it with a vanilla flavor, and then weighing,injecting, and packing the resultant to obtain the desired product. Theproduct is a high quality custard cream which has a smooth gloss, asatisfactory flavor and taste, and well-inhibited retrogradation ofstarch.

EXAMPLE B-8 Chocolate

Forty parts by weight of a cacao paste, 10 parts by weight of a cacaobutter, and 50 parts by weight of a crystalline cyclotetrasaccharidemonohydrate, obtained by the method in Experiment 24 were mixed, and themixture was fed to a refiner to lower the granule size and then placedin a conche for kneading at 50° C. over two days and nights. During theprocessing, 0.5 part by weight of lecithin was added to and welldispersed in the kneaded mixture. Thereafter, the resulting mixture wasadjusted to 31° C. using a thermo controller, and then poured into amold just before solidification of butter, deairated, packed, andsolidified by passing through a cooling tunnel kept at 10° C. Thesolidified contents were removed from the mold and packed to obtain thedesired product. The product has substantially no hygroscopicity,satisfactory color, gloss, and internal texture; smoothly melts in themouth; and has a high quality sweetness and a mild taste and flavor.Also the product can be useful as a low cariogenic, low caloricchocolate.

EXAMPLE B-9 Uiro-No-Moto (A Premix of Uiro (Sweet Rice Jelly))

To 90 parts by weight of rice powder were added 20 parts by weight ofcorn starch, 70 parts by weight of anhydrous crystalline maltitol, 50parts by weight of a powder containing cyclotetrasaccharide obtained bythe method in Example A-1, and four parts by weight of pullulan, and theresulting mixture was mixed to homogeneity into a premix ofuiro-no-moto. The premix and adequate amounts of matcha (a green teapowder) and water were kneaded and then placed in a container andsteamed for 60 min to obtain a uiro with matcha. The product has asatisfactory gloss, mouth feel, flavor, and taste, and it can besuitably used as a long shelf-life low caloric uiro in which theretrogradation of starch is well prevented.

EXAMPLE B-10 An (A Bean Jam)

Ten parts by weight of beans as a material in a usual manner were boiledin a usual manner after the addition of water, removed the astringency,lye, and water-soluble impurities to obtain about 21 parts by weight ofraw bean jam in the form of a granule. To the raw bean jam were added 14parts by weight of sucrose, five parts by weight of a syrup containingcyclotetrasaccharide, obtained by the method in Example A-3, and fourparts by weight of water, and the resulting mixture was boiled, admixedwith a small amount of salad oil, and then kneaded up without pastingthe beans to obtain about 35 parts by weight of the desired product, an.Since the product has a satisfactory stability exhibit syneresis andexcessive color upon baking, it can be arbitrarily used as a materialfor confectioneries such as a bean jam bun, “manju” (a kind of Japaneseconfectionery with bean jam), bean-jam-filled wafer, and icecream/candy.

EXAMPLE B-11 Bread

One hundred parts by weight of wheat flour, two parts by weight of ayeast, five parts by weight of sucrose, one part by weight of a powdercontaining cyclotetrasaccharide obtained by the method in Example A-1,and 0.1 part by weight of a yeast food, were kneaded with water in ausual manner, fermented at 26° C. for two hours, aged for 30 min, andthen baked up. The product is a high quality bread having satisfactorycolor and texture, and adequate elasticity and mild sweetness.

EXAMPLE B-12 Ham

To one thousand parts by weight of ham meat slices were added and groundto homogeneity 15 parts by weight of salt and three parts by weight ofpotassium nitrate, and the resultant slices were piled and allowed tostand over a day and night in a cold-storage room. Thereafter, theresultant slices were first soaked for seven days in a cold-storage roomin a salt solution consisting of 500 parts by weight of water, 100 partsby weight of salt, three parts by weight potassium nitrate, 40 parts byweight of a powder containing cyclotetrasaccharide, penta- orhexa-hydrate, obtained by the method in Example A-5, and an adequateamount of a spice, then washed with cold water in a usual manner, tiedup with a string, smoked, cooked, cooled, and packaged to obtain thedesired product.

The product is a high quality ham having a satisfactory hue, flavor, andtaste.

EXAMPLE B-13 Powdery Peptide

One part by weight of 40% of “HINUTE S”, a peptide solution of ediblesoy beans commercialized by Fuji Oil Co., Ltd., Tokyo, Japan, was mixedwith two parts by weight of a powder containing cyclotetrasaccharide,hepta- or hexa-hydrate, obtained by the method in Example A-6, and theresultant mixture was placed in a plastic vessel, dried in vacuo at 50°C., and pulverized to obtain a powdery peptide. The product having asatisfactory flavor and taste can be arbitrary used as a material forconfectioneries such as premixes, sherbets and ice creams, as well as asubstantially non-digestible edible fiber and a material for controllingintestinal conditions which are used for fluid diets for oraladministration and intubation feeding.

EXAMPLE B-14 Powdery Egg Yolk

Egg yolks prepared from fresh eggs were sterilized at 60-64° C. by aplate heater, and one part by weight of the resultant liquid was mixedwith four parts by weight of a powder containing anhydrous crystallinecyclotetrasaccharide powder, obtained in accordance with the method inExperiment 25. The resultant mixture was transferred to a vessel andallowed to stand overnight to form a block while thecyclotetrasaccharide was allowing to convert into crystallinecyclotetrasaccharide, hepta- or hexa-hydrate. The block thus obtainedwas pulverized by a cutter into a powdery egg yolk.

The product can be arbitrary used as a material for low caloricconfectioneries for premixes, sherbets, ice creams, and emulsifiers, aswell as a substantially non-digestible edible fiber and a material forcontrolling intestinal conditions which are used for fluid diets fororal administration and intubation feeding. Also the product can bearbitrarily used as a skin-beautifying agent, hair restorer, etc.

EXAMPLE B-15 Bath Salt

One part by weight of a peel juice of “yuzu” (a Chinese lemon) wasadmixed with 10 parts by weight of a powder containing anhydrouscrystalline cyclotetrasaccharide obtained in accordance with the methodin Experiment 25, followed by crystallizing to form crystallinecyclotetrasaccharide, hepta- or hexa-hydrate, ageing the formed crystal,and pulverizing the aged crystal to obtain a powder of crystallinecyclotetrasaccharide, hepta- or hexa-hydrate, containing a yuzu peelextract.

A bath salt was obtained by mixing five parts by weight of the abovepowder with 90 parts by weight of grilled salt, two parts by weight ofhydrous crystalline trehalose, one part by weight of silicic anhydride,and 0.5 part by weight of “αG HESPERIDIN”, α-glucosyl hesperidincommercialized by Hayashibara Shoji, Inc., Okayama, Japan.

The product is a high quality bath salt enriched with yuzu flavor andused by diluting in hot water by 100-10,000 folds, and it moisturizesand smooths the skin and does not make one feel cold after taking a baththerewith.

EXAMPLE B-16 Cosmetic Cream

Two parts by weight of polyoxyethylene glycol monostearate, five partsby weight of glyceryl monostearate, self-emulsifying, two parts byweight of a powder of crystalline cyclotetrasaccharide, hepta- orhexa-hydrate, obtained by the method in Example A-8, one part by weightof “αG RUTIN”, α-glucosyl rutin commercialized by Hayashibara Shoji,Inc., Okayama, Japan, one part by weight of liquid petrolatum, 10 partsby weight of glyceryl tri-2-ethylhexanoate, and an adequate amount of anantiseptic were dissolved by heating in a usual manner. The resultantsolution was admixed with two parts by weight of L-lactic acid, fiveparts by weight of 1,3-butylene glycol, and 66 parts by weight ofrefined water, and the resultant mixture was emulsified by a homogenizerand admixed with an adequate amount of a flavor while stirring to obtaina cosmetic cream. The product exhibits an antioxidant activity and has arelatively high stability, and these render it advantageously useful asa high quality sunscreen, skin-refining agent, and skin-whitening agent.

EXAMPLE B-17 Toothpaste

A toothpaste was obtained by mixing 45 parts by weight of calciumsecondary phosphate, 1.5 parts by weight of sodium lauryl sulfate, 25parts by weight of glycerine, 0.5 part by weight of polyoxyethylenesorbitan laurate, 15 parts by weight of a syrup containingcyclotetrasaccharide obtained by the method in Example A-2, 0.02 part byweight of saccharine, 0.05 part by weight of an antiseptic, and 13 partsby weight of water. The product has an improved after taste andsatisfactory feeling after use without lowering the washing power of thesurfactant.

EXAMPLE B-18 Solid Preparation for Fluid Diet

One hundred parts by weight of a power of crystallinecyclotetrasaccharide, penta- or hexa-hydrate, obtained by the method inExample A-6, 200 parts by weight of hydrous crystalline trehalose, 200parts by weight of high maltotetraose content powder, 270 parts byweight of an egg yolk powder, 209 parts by weight of a skim milk powder,4.4 parts by weight of sodium chloride, 1.8 parts by weight of potassiumchloride, four parts by weight of magnesium sulfate, 0.01 part by weightof thiamine, 0.1 part by weight of sodium L-ascorbate, 0.6 part byweight of vitamin E acetate, and 0.04 part by weight of nicotinamidewere mixed. Twenty-five grams aliquots of the resulting composition wereinjected into moisture-proof laminated small bags which were then heatsealed to obtain the desired product.

The product is a fluid diet which is enriched with substantiallynon-digestible edible fiber due to cyclotetrasaccharide, and has asatisfactory intestinal-controlling action. One bag of the product isdissolved in about 150-300 ml of water into a fluid diet and arbitrarilyused by administering orally or intubationally into nasal cavity,stomach, intestines, etc., to supplement energy to living bodies.

EXAMPLE B-19 Tablet

To 50 parts by weight of aspirin were sufficiently admixed with 14 partsby weight of a powder of crystalline cyclotetrasaccharide, penta- orhexa-hydrate, obtained by the method in Example A-7, and four parts byweight of corn starch. The resulting mixture was in a usual mannertabletted by a tabletting machine to obtain a tablet, 680 mg each, 5.25mm in thickness.

The tablet, processed using the filler-imparting ability ofcyclotetrasaccharide, has substantially no hygroscopicity, a sufficientphysical strength, and a quite satisfactory degradability in water.

EXAMPLE B-20 Sugar Coated Tablet

A crude tablet as a core, 150 mg weight, was sugar coated with a firstsolution consisting of 40 parts by weight of a powder of crystallinecyclotetrasaccharide, penta- or hexa-hydrate, obtained by the method inExample A-7, two parts by weight of pullulan having an average molecularweight of 200,000, 30 parts by weight of water, 25 parts by weight oftalc, and three parts by weight of titanium oxide until the total weightreached to about 230 mg. The resultant was then sugar coated with asecond solution consisting of 65 parts by weight of a fresh preparationof the same powder of crystalline cyclotetrasaccharide, penta- orhexa-hydrate, one part by weight of pullulan, and 34 parts by weight ofwater, and glossed with a liquid wax to obtain a sugar coated tablethaving a satisfactory gloss and appearance. The product has a relativelyhigh shock tolerance and retains its high quality for a relatively-longperiod of time.

EXAMPLE B-21 Ointment for Treating Trauma

To 100 parts by weight of a powder of crystalline cyclotetrasaccharide,penta- or hexa-hydrate, obtained by the method in Example A-7, and 300parts by weight of maltose was added 50 parts by weight of methanoldissolving three parts by weight of iodine, and further added 200 partsby weight of a 10% (w/v) aqueous pullulan solution to obtain the desiredproduct with an adequate extensibility and adhesiveness. The product isa high-valued ointment in which the dispersion of iodine and methanol iswell inhibited by cyclotetrasaccharide and is relatively low in changeduring storing.

Because the product exerts a sterilizing action by iodine and acts,based on maltose, as an energy-supplementing agent to living cells, itshortens the curing term and well cures the affected parts and surfaces.

INDUSTRIAL APPLICABILITY

As described above, the present invention relates to a novelα-isomaltosylglucosaccharide-forming enzyme, and their process and uses,more particularly, to a novel α-isomaltosylglucosaccharide-formingenzyme, process thereof, microorganisms producing the enzyme,α-glucosyl-transferring method using the enzyme, a method for formingα-isomaltosylglucosaccharide, a process for producingcyclotetrasaccharide having the structure ofcyclo{66)-α-D-glucopyranosyl-(163)-α-D-glucopyranosyl-(166)-α-D-glucopyranosyl-(163)-α-D-glucopyranosyl-(16},and a composition comprising the saccharide obtainable therewith.According to the present invention, an industrially usefulcyclotetrasaccharide having the structure ofcyclo{66)-α-D-glucopyranosyl-(163)-α-D-glucopyranosyl-(166)-α-D-glucopyranosyl-(163)-α-D-glucopyranosyl-(16}or a composition comprising the same can be produced on an industrialscale and at a relatively low cost. Since these cyclotetrasaccharidesand the saccharide comprising the same have substantially no or lowreducibility, substantially do not cause the amino carbonyl reaction,substantially do not exhibit hygroscopicity, have easily handleability,have mild sweetness, adequate viscosity, moisture-retaining ability,inclusion ability, and substantially no digestibility, they can beadvantageously used in a variety of compositions such as food products,cosmetics, pharmaceuticals as a sweetener, material for low caloricfoods, taste-improving agent, flavor-improving ability,quality-improving agent, syneresis-preventing agent, stabilizer, filler,inclusion agent, and base for pulverization. The present invention,having these outstanding functions and effects, is a significantlyimportant invention that greatly contributes to this art.

1. A process for producing a cyclotetrasaccharide having the structureofcyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→}or a saccharide composition comprising the same, which comprises thesteps of: (a) allowing an α-isomaltosylglucosaccharide-forming enzyme toact on a solution containing saccharide having a glucose polymerizationdegree of at least two and α-1,4 glucosidic linkage as a linkage at thenon-reducing end of the saccharide to form α-isomaltosylglucosaccharidehaving a glucose polymerization degree of at least three, an α-1,6glucosidic linkage as a linkage at the non-reducing end, and α-1,4glucosidic linkages at all positions except the linkage at thenon-reducing end; whereby said α-isomaltosylglucosaccharide-formingenzyme forms said α-isomaltosylglucosaccharide from said saccharide bycatalyzing an α-glucosyl-transferring reaction; (b) allowing anα-isomaltosyl-transferring enzyme to act on the formedα-isomaltosylglucosaccharide to form said cyclotetrasaccharide; wherebysaid α-isomaltosyl-transferring enzyme forms said cyclotetrasaccharidefrom said α-isomaltosylglucosaccharide; and (c) collecting the formedcyclotetrasaccharide or a saccharide composition comprising the samefrom the resulting reaction mixture.
 2. The process of claim 1, whereinsaid saccharide having a glucose polymerization degree of at least twoand α-1,4 glucosidic linkage as a linkage at the non-reducing end is oneselected from the group consisting of maltooligosaccharides,maltodextrins, amylodextrins, amyloses, amylopectins, soluble starch,gelatinized or liquefied starches, and glycogens.
 3. The process ofclaim 2, wherein said gelatinized or liquefied starches has a DE(dextrose equivalent) of not higher than
 20. 4. The process of claim 1,wherein in steps (a) and (b), cyclomaltodextrin glucanotransferaseand/or starch debranching enzyme are allowed to act on the solutioncontaining the saccharide simultaneously with steps (a) and (b).
 5. Theprocess of claim 1, wherein after steps (a) and (b), one or more enzymesselected from the group consisting of α-amylase, β-amylase, glucoamylaseand α-glucosidase are allowed to act on the resulting mixture.
 6. Theprocess of claim 1, wherein in step (C), the product is purified byconducting one or more purification methods selected from the groupconsisting of decoloration, desalting, fractionation by columnchromatography, separation with a membrane, fermentation treatment usingmicroorganisms, and decomposition by an alkaline treatment.
 7. Theprocess of claim 1, wherein said reaction mixture contains at least 30%(w/w), on a dry solid basis, of the cyclotetrasaccharide.
 8. The processof claim 1, wherein said cyclotetrasaccharide or said saccharidecomposition comprising the same is in the form of a syrup, massecuite,amorphous powder, amorphous solid, crystalline powder, or crystallinesolid.
 9. The process of claim 8, wherein said crystal is prepared bycrystallizing in an aqueous solution without using any organic solvent.10. The process of claim 1, wherein saidα-isolatosylglucosaccharide-forming enzyme has the followingphysicochemical properties: (1) Molecular weight 140,000±20,000 daltons,137,000±20,000 daltons, 136,000±20,000 daltons, or 94,000±20,000daltons, when determined on SDS-PAGE; (2) Optimum temperature About 40°C. to 50° C. when reacted at a pH of 6.0 for 60 min, or about 45° C. to55° C. when reacted in the presence of 1 mM Ca²⁺; or About 60° C. whenreacted at a pH of pH 8.4 for 60 min, or about 65° C. when reacted inthe presence of 1 mM Ca²⁺; (3) Optimum pH About pH 6.0 to 6.5 or pH 8.4when reacted at 35° C. for 60 min; (4) Thermal stability Stable up to atemperature of about 35° C. to 45° C., or of about 40° C. to about 50°C. in the presence of 1 mM Ca²⁺ when incubated at pH 6.0 or 8.0 for 60min; or Stable up to a temperature of about 60° C. or of about 65° C. inthe presence of 1 mM Ca²⁺ when incubated at pH 8.0 for 60 min; (5) pHstability Stable at a pH of about 4.5 to about 10.0 when incubated at 4°C. for 24 hours.
 11. The process of claim 1, wherein saidα-isomaltosyl-transferring enzyme has the following physicochemicalproperties: (1) Molecular weight 112,000±20,000 daltons, 102,000±20,000daltons, or 116,000±20,000 daltons when determined on SDS-PAGE; (2)Optimum temperature About 40° C. to 50° C. when incubated at a pH of 6.0for 30 min; (4) Optimum pH About 5.5 to 6.5 when incubated at 35° C. for30 min; (5) Thermal stability Stable up to a temperature of about 40° C.to 45° C. when incubated at pH 6.0 for 60 min; (6) pH stability Stableat a pH of about 3.6 to 10.0 when incubated at 4° C. for 24 hours. 12.The process of claim 1, wherein saidα-isomaltosylglucosaccharide-forming enzyme and saidα-isomaltosyl-transferring enzyme are obtained from a microorganism ofthe genus Bacillus or Arthrobacter.
 13. The process of claim 12, whereinsaid microorganism of the genus Bacillus is one selected from the groupconsisting of Bacillus globisporus C9, FERM BP-7143; Bacillusglobisporus C11, FERM BP-7144; Bacillus globisporus N75, FERM BP-7591;and mutants thereof.
 14. The process of claim 12, wherein saidmicroorganism of the genus Arthrobacter is one selected from the groupconsisting of Arthrobacter globiformis A19, FERM BP-7590; Arthrobacterramosus S1, FERM BP-7592; and mutants thereof.